Pinnacle-Manual Apr2016 Web2

Operator Manual
Post-Column
Derivatization
AP
TEE
PR
OV
ED
RY
AN
HE
MIS
1280 Space Park Way

Mountain View
CA 94043
USA
GUA
Instrument
800-654-3330 650-694-6700
650-968-0749
fax
www.pickeringlabs.com
support@pickeringlabs.com
http://pickeringlabs.blogspot.com
Cat. No. 0101-0008
Version 10, April 2016

Copyright 2005 by Pickering Laboratories, Inc. All rights reserved. No Portion of this manual or
any artwork contained herein may be reproduced in any shape or form without the
express written consent of Pickering Laboratories, Inc.
Table of Contents

Getting Started
How to Use this Manual
Read this First! 2
Symbols and Warnings
Specifications 2
Site Requirements 4
1.1
Section 1
Introduction
What is Post-column Derivatization? 1.1

Requirements for a Successful Post-column Method
Design of a HPLC system 1.2
Designing a Post-column System 1.4
Design of the Pinnacle PCX 1.6
2.1
Section 2
1.1
Overview
Introduction 2.2
Check Valve 2.3
Column Connections and Column Oven 2.3
Reagent Pump 2.4
Reagent Valves 2.5
Reservoir Tray / Bottles 2.5
Fluidics Panel 2.7
Quick Change Reactor 2.8
Detector Connections 2.8
Pump Compartment 2.9
Gas Manifold 2.9
Display Module 2.10
Electronics Compartment 2.11
Back Panel 2.11
Safety Features in the Pinnacle PCX 2.13
Standard Configurations of Pinnacle PCX 2.14
Pinnacle Operators Manual

Pickering Laboratories Inc.
II
3.1 Section 3
Installation
Site Requirements 3.2

Instrument Unpacking and Preparation 3.4
Unpacking 3.4
Gas Connections to Back Panel of Pinnacle PCX
Computer Connections 3.6
Software Installation 3.6
3.5
Pump and Autosampler Connections 3.8

Eluant Priming3.10
Column and Guard Installation 3.10
Detector Connections 3.11
Reservoir Connections 3.12
Reagent Pump Preparation 3.13
Running a Chromatogram 3.14
Shutdown 3.15
4.1
Section 4
Pinnacle PCX Operation
Software Overview4.1
Status Window 4.2
Menu Options/Functions 4.4

File

Control

Method
Sequence

Instrument
Configuring your Pinnacle PCX 4.6
Log Files 4.7
Creating and Editing Methods 4.7
Creating and Editing Sequences 4.10
Tutorials 4.11
Running post-column analysis using Pinnacle PCX
STOP/RESET Pinnacle PCX 4.17
Pinnacle Shutdown 4.17

4.14
III
5.1 Section 5
Maintenance
Maintenance Suggestions 5.1

Operating Suggestions 5.2
Proper Shutdown Procedures 5.3
Basic Maintenance Procedures 5.6
Reagent Filter(s) Replacement 5.7
Ambient Reactor Replacement 5.8
Replacement of Heated Reactor Cartridge5.8
Valve Maintenance 5.9
Pump Seal Replacement 5.12
Fuse Replacement 5.16
Tubing Kit Guides 5.17
6.1 Section 6
Applications
Amino Acids 6.2

Introduction 6.2

Background 6.2-1

Basic Sample Preparation 6.2-2

Reagent Preparation 6.2-3
Post-Column Conditions 6.2-4

Procedure 6.2-5

Sample Chromatograms and Gradient Programs

Precautions 6.2-18
Carbamates 6.3

Introduction 6.3

Background 6.3



Post-Column Conditions 6.3-5

Analytical Procedure 6.3-5


Precautions 6.3-12
6.2-6
6.3-5

IV
Glyphosate 6.4

Introduction 6.4

Background 6.4-1



Analytical and Post-Column Conditions

Analytical Procedure 6.4-4

Sample Chromatograms 6.4-5

Precautions 6.4-6
7.1 Section 7
6.4-4
Troubleshooting
Contact Pickering Laboratories For Support

Instrument Parameter Log 7.2
General Troubleshooting Advice 7.2
Common System Problems 7.3
Common Chromatography Problems 7.4
Common Column Problems 7.6
Application Specific Troubleshooting 7.7

Amino Acids

Carbamates

Glyphosate
Amino Acid7.7
Software Troubleshooting 7.8
7.1
Procedures7.11

To Remove Silica Deposits From Reactor

To Remove Mineral Deposits In The Reactor From Hard Water

To Remove Grease Deposits

If Reagent Backflows Onto Column

If TRIONE Backflows Onto Column

If NaOH Is On Column

To Remove Iron Contamination From Column

To Pump RESTORE Through The Glyphosate Column

8.1 Appendices
Installation/Operational Qualitication of the Pinnacle PCX 8.2
Installation/Operational Qualitication of the Pinnacle PCX Data Sheet
Performance Qualifications of the Pinnacle PCX Data Sheet 8.8
Sample Instrument Parameter Log 8.15
Flow Diagram Simplex 8.16
Flow Diagram Duplex8.17
Parts List for Pinnacle PCX 8.18
Recommended Consumables and Spare Parts
Limited Warranty 8.24
References 8.25
8.7
8.21
9.1 Index

VI

G etting S tarted
Getting Started
1

2 Read this First!
2 Symbols and Warnings
2 Specifications
4 Site Requirements

The Pinnacle PCX manual is designed to contain all of the information necessary for the installation, operation,
maintenance and troubleshooting of the Pinnacle PCX. It is designed to contain any and all information that you
may require during the lifetime of your Pinnacle PCX.
In addition to the general operation information, this manual also contains sections devoted to a particular
application. Since Pickering Laboratories provides the complete solution, we included the chromatograms,
operating conditions, and some troubleshooting information for our most commonly supported methods.
As well as instructional information, this manual also contains a section for recording service information such
as serial numbers, installation dates, service dates etc.
Each section of the manual is divided by a tab with the title of that section. For easy reference, simply select
the tab that you require based on the information you are looking for. For example, if you are running Amino
Acids, and have a question about the gradient program, simply go to the section titled Amino Acids.

G etting S tarted
Read this First!

Before attempting to install the Pinnacle PCX post-column derivatization instrument, it is vitally important that
you read this manual first, and attend to site, HPLC, and accessories requirements:
HPLC Page 5
Gas Supply Requirements Page 5
Reagent Reservoir bottles Page 5
Computer Page 5
Symbols and Warnings
Caution this symbol indicates that caution must be used when dealing with this part.
Hot this symbol is located on the Heated reactor, which can reach scalding temperatures.
Specifications
INSTRUMENT
Dimentions
21.50 H x 10.63 W x 18.25 D inches (54.0 x 26.7 x 46.4 cm), instrument only, doors closed
Weight
77lbs for Dual-pump systems
67lbs for Single-pump systems
Reagent Pumps
Max operating pressure 500 psi (35bar)
Flow rate range 50l 1500l/minute
Refill cycle 60 seconds
Heated Reactor
5C above ambient to 130C
Thermal Safety switch limits temperature to 150C
Stability +/- 0.5C
Accuracy +/- 1C

G etting S tarted
Electrical
720 W
120 VAC +/- 10%, 240 VAC +/- 10%
5 A maximum at 108 VAC
47 63 Hz
Installation over voltage category: Pinnacle PCX complies with Class B Emission Test Specifications.
Fuses
2 ea, 5mm x 20 mm, 6 A, time lag
LCD
Backlit, positive mode, high contrast, viewing area 125mm (L) x 75mm (W)
COMMUNICATION
USB
Requires a USB port.
Relay
Any machine that drives this relay input shall provide a relay contact pair that is electrically isolated from all
other electrical devices. The relay contacts must be capable of switching 1mA at 24 +/- 2 Vdc.
Ethernet (optional)Connector: RJ45
Connection Speed: 10/100-BASE-T
Ethernet Protocol: TCP/IP
IP Address: User set or DHCP (DHCP requires the Network Administrator to Reserve an IP address with the
DHCP server)
Note: Recommend connection to a Network Switch or Router, or direct connection to a dedicated Network
card in the PC.
ENVIRONMENTAL
Indoor use only
Altitude up to 6500 ft (1981 m)
Ambient Temperature 40C
Relative Humidity up to 80% at 31C

G etting S tarted
Radio Frequency Pinnacle PCX complies with IEC 61000-4-3

This device complies with Part 15 CHECK Class B of the FCC Rules. Operation is subject to the following
two conditions: (1) This device may not cause harmful interference, and (2) this device must accept any
interference received, including interference that may cause undesired operation.
WETTED MATERIALS
PEEK, PTFE, Air Barrier Tubing
99.9% Ceramic
Perlast
Borosilicate glass
EPR
SOFTWARE
PCX Control Software runs on Microsoft Windows XP, Vista, or Windows 7.
10Mb hard disk space
PCX History Log file: Maximum file Size on the computer: 1.2 Mb
Site Requirements
INSTRUMENT
Bench Space
33 H x 17 W x 21 D inches (84 x 56 x 59 cm), both doors fully opened, with bottles and electrical connections
in place.
Minimum 3 inches clearance at back of instrument for venting.

Note: Space quoted above is for Pinnacle PCX.
The total space requirement depends on the brand and model of HPLC.
Electrical Outlet
One grounded outlet must be provided for the Pinnacle PCX.

G etting S tarted
Computer
IBM-compatible
PCX Control Software runs on Microsoft Windows XP, Vista, or Windows 7.
Ethernet or USB port
Available Memory: Minimum 10Mb
Ethernet (optional)Connector: RJ45
Connection Speed: 10/100-BASE-T
Ethernet Protocol: TCP/IP
IP Address: User set or DHCP (DHCP requires the Network Administrator to Reserve an IP address with the
DHCP server)
Note: Recommend connection to a Network Switch or Router, or direct connection to a dedicated Network
card in the PC.
Relay
For synchronization, the HPLC system must be capable of sending a relay signal to an external instrument.
No relay connection is needed for Agilent 1100 or Agilent 1200. Pinnacle PCX software directly synchronizes
with Chemstation version 9.0 or higher.
Gas Supply
High purity Nitrogen, 45-75 psi (min - max)
Outlet of regulator must connect to 1/8 OD tubing
Reagent Reservoir Bottles
The Pinnacle PCX includes one pressurized reagent reservoir for the one reagent system and two for the two
reagent system.
Note: For your safety, the bottles are coated with a tough plastic film and rated to a maximum of 15 psig
(1 bar). Do not use uncoated bottles.
HPLC Pump
Binary gradient for glyphosate, carbamate applications.
Quaternary for all others.

G etting S tarted
Autosampler
Minimum injection volume 10l, preferably by full-loop injection
For drinking water, minimum injection volume 200l
Tefzel rotor seal required for all applications using eluants with pH>10
PEEK needle seat required for all applications using eluants with pH>10
Detector
Pressure rating of flow cell must be > 110 psi
Inlet capillary must be > 0.17 mm ID
MISCELLANEOUS SUPPLIES
For Amino Acid analysis, a Dead-Head kit is required. This can be purchased from Pickering Laboratories.
Chemistry
The user must check the chemistry requirements for the specific application.
For Carbamate Analysis
HPLC Grade Methanol
HPLC Grade Water
Materials for calibration standards
Carbamate hydrolysis reagent (Cat. No. CB910)
Carbamate OPA diluent (Cat. No. CB130 or CB130.2)
o-phthaladehyde (Cat. No. O120)
Thiofluor (Cat. No. 3700-2000)
For Glyphosate Analysis
5% Sodium hypochlorite solution
Materials for calibration standards
Methanol for OPA reagent preparation
Glyphosate Eluant, pH2.0 (Cat. No. K200)
Glyphosate Regenerate (Cat. No. RG019)
Glyphosate Hypochlorite diluent (Cat. No. GA116)
Glyphosate OPA diluent (Cat. No. GA104)
o-phthaladehyde (Cat. No. 0120)
Thiofluor (Cat. No. 3700-2000)

G etting S tarted
For Amino Acid Analysis

A: Fluorescence detection
Methanol for OPA reagent preparation
5% Sodium hypochlorite if using the 2-reagent method
Brij 35 solution for OPA reagent preparation
DI Water
Pickering sodium or lithium elution buffers
(see application section for cat. nos.)
B: UV Visible detection
DI Water
TRIONE Ninhydrin reagent (cat. no. T1OOC or T200)
Pickering sodium or lithium elution buffers
(see application section for cat. nos.)

G etting S tarted
Notes

1.1
Section 1
I ntroduction
Section 1
Introduction
1.1 What is Post-column derivatization?
1.1 Requirements for a Successful Post-column Method
1.2 Design of a HPLC system
1.4 Designing a Post-column system
1.6 Design of the Pinnacle PCX
What is Post-column Derivatization?

This is a method which renders visible certain compounds that are normally invisible. Since this reaction
occurs after, or post-separation, it is referred to as post-column deriviatization. The analytes of interest are
separated on the column first, and then reacted with a chemical that will render them detectable at a desirable
wavelength, voltage, or any number of various means of detection.
Post column derivatization enhances the sensitivity of HPLC by several means:
1) Most reagents are selective for a particular class of substances, so analytes of that class are more easily seen
against a complex background.
2) Since the separation is performed first, the matrix of the sample is either washed off of the column before
the analytes, or is retained by the column. This leaves a very pure sample of analyte to react. This eliminates
the need for extensive sample clean-up, and provides a very reproducible reaction because there are no matrix
interferences.
The Pinnacle PCX post-column derivatization instrument automatically mixes the stream of effluent flowing
from the HPLC column with a stream of reagent solution. The mixture flows through a reactor to allow enough
time for the chemical reactions to complete. In many cases, the reaction is very slow at room temperature. For
this reason, the reactor can be heated. There are some methods that require two or more reagents added in
sequence. This is done by the addition of a second reagent pump. In many cases, the second reaction occurs
at a much faster rate, and can be efficiently accomplished at room temperature. After the reaction is complete,
the derivatives flow into the detector, where the absorbance or the fluorescence (usually) is measured by the
HPLC system. These two means of detection are the most common, but they are certainly not the only means of
detection.
Requirements for a Successful Post-column Method

There are many things to take into consideration when developing a method and instrument for postcolumn derivatization. For example, many pumps have a periodic motion when drawing and dispensing that

1.2
Section 1
I ntroduction
will manifest itself in the baseline of a chromatogram unless it is properly dampened. Below are the basic
requirements for a successful automated post-column method:
1) Reagent Stability. The minimum reagent stability sufficient for routine work is one day. This means that
the yield and signal-to-noise ratio for a given sample must remain constant for at least 8 hours.
2) Reaction Speed. The analytical separation is complete when the reagent is mixed with the column effluent.
Therefore it is important that the analyte react as quickly as possible. The longer the reaction time, the larger
the reactor volume required. With larger volumes, the peak shape will become distorted. To minimize band
spreading, it is important to keep the overall time (and therefore volume) as low as possible between the
column and detector. If the reaction is slow (in excess of one minute), an elevated temperature can be used to
decrease the reaction time.
3) Reproducibility. Because the reaction is occurring on the fly, as the combined column and reagent
stream flows toward the detector, the reproducibility is linked to the flow rate precision of the pumps and to the
temperature. Accordingly, even an incomplete reaction will be as repeatable as the retention time for any given
species. Therefore, it is important that the pumps maintain a constant flow rate, and that the reactor maintain a
constant temperature. It is also very important that the column be maintained at constant temperature to ensure
that the analytes are properly separated and identified.
4) Minimal Detector Response of Reagents. The color or background fluorescence of the reagent (or
its by-products) represents a continuous noise source. Because the reagent is present in excess relative to the
analyte, the analytes signal could be obliterated by the reagents strong background signal. The baseline noise
is proportional to the background signal.
5) Solubility. All species must remain in solution, including the combined components of the eluants and the
reagent(s), as well as the newly formed derivative(s). Precipitates can block capillary tubes, burst reactors, and
foul detector flow cells.
6) Uniformity of flow. The baseline noise is a function of the flow-noise in the eluant and reagent pumps.
Non-uniform flow causes non-uniform mixing leading to modulation of the background signal which appears
as noise. Refractive index noise can be even more objectionable than absorbance noise. Common techniques
for evening the flow of the pumps is the addition of a pulse dampener, or the use of a syringe pump.
Design of a HPLC system

This next section is a simplified view of a HPLC, followed by the ideas behind a post-column system. This
section is intended to help novice HPLC operators.

1.3
Section 1
I ntroduction
In order to understand post-column HPLC, we need to understand the design of an HPLC. If we connect an
HPLC pump directly to a detector (with nothing in between), the baseline from the detector shows a periodic
noise (Figure 1-1); the time period is equivalent to the pump stroke.
Injector
Detector
LC Pump
Baseline
Waste
Eluants
FIGURE 1-1
Now add a commercial pulse dampener. The baseline is still not smooth; the periodic noise is still there
although less pronounced (Figure 1- 2). The pulse dampener absorbs most of the pulses from the pump, but
the flow requires more stabilization.
Injector
Commmercial
Pulse
Dampener
(capacitor)
LC Pump
Detector
Baseline
Waste
Eluants
FIGURE 1-2
A restriction inline will cause the flow at the outlet of the restriction to be constant. In an HPLC system, this is
accomplished with the analytical column. Actually, the column does more than separation; it creates a backpressure. It is the combination of the pulse dampener and the column that creates a smooth baseline.
(Figure 1-3)

1.4
Section 1
I ntroduction
Injector
Pre-column
Check Valve
Pulse
Damper
LC Pump
Detector
Thermostatted
Column & Guard
Baseline
Waste
Eluants
FIGURE 1-3
An analogy will help us understand the concept. Let us use a river as an example. If it rains; the river swells.
If it stops raining; the level goes down. As the level fluctuates, it is equivalent to a periodic noise. To obtain a
constant flow, we need to add a reservoir (pulse dampener) and a dam (column). The flow downstream from
the dam is constant (smooth baseline).
Designing a Post-column System

The same principals that are used in HPLC can be applied to the post-column system. What happens if we
simply add a post-column pump, a mixing tee, and a reactor? The periodic noise returns to the baseline
(created by the post-column pump; Figure 1-4).

1.5
Section 1
I ntroduction
Injector
Filter
Thermostatted
Column & Guard
Heated
Reactor
Pulse
Damper
Detector
Back-pressure
Regulator
LC Pump
Eluants
Waste
Baseline
Post-column
Pump
Reagent
Bottle
FIGURE 1-4
However we do not need to invent anything new; we just need a pulse dampener and a column. In the
generation of Post-column systems prior to the Pinnacle PCX, the restriction performed by the column was
achieved using a Restrictor, which is packed with very inert material. With this flow conditioner in place, the
baseline is now acceptable.

1.6
Section 1
I ntroduction
Design of the Pinnacle PCX

The Pinnacle PCX has taken the post-column system to a new level. The introduction of a syringe pump has
eliminated the need for a pulse-dampener and restrictor by:
The Pickering syringe pumps complete a filling cycle prior to the injection of a run, and deliver reagent
during the run at a constant rate.
There is a valve between the pump and reactor which also helps to regulate the reagent flow, by opening key
ports at the appropriate time.
There is also a pressure transducer which we have added in line before the valve to determine if there are
any blockages in the reactors.
Now you are ready for Section 2.

2.1
Section 2
O verview
Section 2
Overview
2.2 Introduction
2.3
2.3
2.4
2.5
2.5
2.7
2.8
2.8
2.9
2.9
2.10
2.11
2.11
2.13
2.14
Check Valve
Column Connections and Column Oven
Reagent Pump
Reagent Valves
Reservoir Tray / Bottles
Fluidics Panel
Quick Change Reactor
Detector Connections
Pump Compartment
Gas Manifold
Display Module
Electronics Compartment
Back Panel
Safety Features in the Pinnacle PCX
Standard Configurations of Pinnacle PCX

2.2
Section 2
2
COLUMN
PUMP 2
3 VALVE 2 5
4
FILTER
AMBIENT
REACTOR
HEATED
REACTOR
In addition to accomplishing the above three tasks, the

Pinnacle PCX has various features to make the analysis
more reliable, convenient, and simple. It also contains
features to protect the instrument from accidental
damage.
TRANSDUCER 1
FILTER
FLUSH
PUMP 1
R1
R2
TRANSDUCER 2
This chapter is designed to familiarize you with the

components, layout, and function of the Pinnacle PCX.
Here you will find descriptions of each key component
of the instrument and what it does.
At the most basic level, the Pinnacle PCX performs
three main tasks:
1 .Regulates the temperature of the analytical column
2. Delivers the reagent
3. Heats the reaction
R1
Introduction
FLUSH
O verview
VALVE 1 5
4
WASTE
FROM
HPLC
Flow Path Dual Pump
TRANSDUCER
HEATED
REACTOR
3
PUMP
VALVE 1 5
4
WASTE
FROM
HPLC
Flow Path Single Pump

Figure 2-2
The flow path of the Pinnacle PCX is extremely inert,

rendering it very versatile. The same instrument can be
used for many different applications, and will tolerate a
high percentage of reagents.
FILTER
COLUMN
TO DETECTOR
TO DETECTOR
Figure 2-1
2.3
Section 2
O verview
Check Valve
Figure 2-3
The column check valve is located just outside of the

column oven (figure 2-3). The check valve is designed
to protect the column against backflow of the reagents in
case of unexpected HPLC shutdown.
Column outlet tubing

Column oven door
Rubber foam seal
Column Connections and Column Oven

The column oven contains the analytical column and
guard column (figure 2-3). The oven is a convection air
oven. The column heater utilizes re-circulating air flow
technology to provide quick, uniform column heating.
Fast column cooling is assisted by the introduction of
fresh air flow into the chamber by means of two fans
that are controlled by the Pinnacle PCX control software.
The temperature range holds within 1C resolution
from 5C above ambient to 75C. A temperature gradient
can be performed on the column to effect an increase
in the speed of the separation. The temperatures can
be programmed for a gradient with as many steps as
required for fine-tuning an analysis.
Analytical column
Inlet end
Coupler/column connector
Guard column
The heater block is designed to receive the analytical

column (5, 10, 15, and 25 cm long) and the guard
column when they are connected via a column easyconnect fitting (included with the instrument, and sold
separately by Pickering, Cat. #3102-3064).
The outlet end of the column hangs in the slot at the top
Check valve
of the column oven. The inlet to the guard column is at
to Guard
the bottom of the column oven door.
from HPLC
Column
The column oven door contains a pocket to fit around
the analytical column and guard. There is a groove in
Blue Peek Tubing PEEK Nut
PEEK Nut
Check Valve
(3101-0011)
(1452-0226)
the door that is lined with Santoprene foam rubber cord
(3106-1007)
that seals the door to the chassis and provides a stable
insulated heating environment.
The last part of the lead-in capillary is located inside the column oven to preheat the eluant for a more uniform
temperature within the column.

2.4
Section 2
O verview
Make sure that the column door is securely closed before starting operation.
Warning. The column heating block may become hotter than 75C. For your safety, wear insulating gloves
when the column oven is warm.
Attention. La rsistance chauffante de la colonne peut dpasser une temprature de 75C. Pour votre scurit,
prire de porter des gants isolants lorsque le four de la colonne est chaud.
Warnung! Der Heizblock des Sulenofens knnte heier als 75C werden. Fr Ihre Sicherheit sollten Sie
isolierende Handschuhe tragen, wenn der Sulenofen warm ist.
Atencin. El bloque calefactor de columnas puede estar por encima de 75C. Para su seguridad use guantes
aislantes cuando el horno de columnas est caliente.
Avvertimento. Il blocco della colonna potr diventare molto caldo e superare ai 75C. Per la sua protezione
usa guanti con insulazione per questa applicazione.
Reagent Pump
The reagent pump is a syringe pump. Refer to figure 2-4 and 2-8 (items 4 and 10). The syringe has a volume
of 70 ml. The flow rate can be programmed from 0.05 ml/min to 1.5 ml/min. Once programmed, the syringe
pump delivers at a constant speed. Since there is no need for constant refilling and dispensing (as with a
reciprocating pump), there is a very high flow precision that does not require external pulse dampening
features.
The syringe pump cylinder and head is made from a single piece of 99.9% Alumina for ruggedness and nonreactivity. The piston surface is made from PEEK with an inert o-ring seal.
The pump contains a piston wash for extended seal life. The piston wash is automatically performed with the
movement of the piston.
For purposes of fast refill and flushing, the pump
can refill or dispense (to waste) at 55 ml/min.
Syringe pump
cylider
Figure 2-4

2.5
Section 2
O verview
Reagent Valves
Reagent Valves are placed immediately after
the pump. The valves have 5 possible operating
positions:
1) REAGENT: From Reagent Bottle to Pump (3-4)
2) REACTOR: From Pump to Reactor (1-2)
3) WASTE: From Pump to Waste (1-6)
4) FLUSH: From Flush Bottle to Pump (5-4)
5) BLOCKED: The valve ports are blocked.
6
Valve
Pump
The valves are connected to a Flush Bottle which

can be used to flush the reactor in the event of a
shutdown or to aid in flushing the system. This will
reduce the likelihood that the reactor becomes
blocked. The valves are also connected to the
reagent bottles and are used for filling the syringe pump.
Figure 2-5
Reservoir Tray / Bottles

This is the tray located on top of the instrument. It holds the
reagent bottles, wash bottle and flush bottle. It is removable
for cleaning. It will hold 1L of liquid and as such is considered
secondary containment. It does not seal any openings at the top
of the instrument in case of heavy spillage.
Flush
Piston
Wash
Reagent
There are 4 bottles located here:

1. Reagent Reservoir 1
2. Reagent Reservoir 2
3. Flush Bottle contains water or 80-20 water/alcohol (IPA or
Methanol) for flushing the instrument.
4. Piston Wash bottle contains 90-10 water/alcohol (IPA or
Methanol). This bottle is connected via 1/4 inch OD flexible
tubing to the piston wash of each pump in the instrument.
Change Wash and Flush solutions at least once a week to prevent
contamination.
The pressurized reagent reservoir serves two purposes:
It protects air-sensitive reagents from oxidation.
It helps the syringe pump fill consistently and quickly by
providing a source of pressure.
Figure 2-6

2.6
Section 2
O verview
Reagent tubing is pre-connected to the pump at the factory. All

Reagents lines are Air Barrier tubing. Reagent 1 is labeled R1,
and Reagent 2 is labeled R2 throughout the instrument. Nuts
and ferrules (1/4-28 ) are provided for connecting the tubing
to the Reservoir caps.
The reservoir cap (Figure 2-7A) has two connecting ports
on the top one for connecting reagent line and one for
connecting the gas line. The port for the gas line has a one-way
valve to prevent backflow of the reagent into the gas manifold in
case of a drop in gas pressure. Use nuts and ferrules (1/4-28)
Figure 2-7A
to make the connections. There are two stopcocks, one is for
the vent port and the other is for the reagent line. Keep reagent line stopcock open for operation. Open the vent
stopcock to sparge the reagent and close it to pressurize the reservoir. This is the normal operating position.

When changing reagent, first turn off the gas using the toggle valve on the fluidics panel. Then vent the reagent
bottle by opening vent stopcock. Now you can safely remove the cap. It is convenient to have extra bottles
so that you can simply transfer the cap without setting it down and risking
contamination.
Valve
If you have a reservoir cap shown on figure 2-7B, the large white knob is the
valve; pull it up for CLOSED, and push it down for OPEN. If the gas is turned on,
opening the vent valve will sparge the reagent. Closing the valve will pressurize the
reservoir; this is the normal operating position. On the side of the cap, away from
the on-off valve, there is a 1/4-28 fitting; you may optionally connect a tube here
to carry vapors to an exhaust vent.
Figure 2-7B
Warning. For your safety, the bottles are coated with a tough plastic film and are
rated to a maximum of 15 psig (1 bar). Do not use uncoated bottles.
Attention. Pour votre scurit, les bouteilles sont recouvertes dun film de plastique dur, et sont calibres un
maximum de 10 psig (0.7 bar). Ne pas utiliser les bouteilles non recouvertes.
Warnung! Fr Ihre Sicherheit wurden die Reagenzienflaschen mit einem festen Schutzberzug aus Kunststoff
versehen. Die Flaschen sind bis max. 0.7 bar (10 psig) zugelassen Flaschen mit beschdigtem Schutzberzug
drfen nicht mehr benutzt werden Verwenden Sie keine Flaschen ohne Schutzberzug!
Atencin. Para su seguridad, las botellas estn recubiertas con una resistente pelcula plstica, y estn
constrastadas a 10 psig (0.7 bar). No utilice botellas sin recubrimiento.
Avvertimento. Per la sua protezione, le bottiglie sono construite forti con un percentuale du plastica, e sono
usabili per un massimo di 0.7 Bar (10 psi). Non usare bottiglie normali.

2.7
Section 2
O verview
There are three types of Reservoir/Cap assemblies used on the Pinnacle PCX:
1) Reagent Reservoir is 1L, 45-430, safety-coated. These are shipped with blue caps with white valve assembly.
These can be pressurized.
2) The wash Reservoir is 1L, 38-430, clear glass. These are shipped with caps with 1ea 1/4 opening and 2ea
1/8 openings. These bottles cannot be pressurized, and do not contain threads for tubing connections.
3) The flush Reservoir is 1L, 38-430, clear glass. These are also shipped with caps, but with 3 each 1/8
openings. These bottles cannot be pressurized, and do not contain threads for tubing connections.
Note: The two types of caps cannot be interchanged because the necks of the bottles are different sizes.
Fluidics Panel
The fluidics panel is the busiest area of the
instrument. Everything that you will need is
located on the front of the panel. The door can
be removed if desired by the user for easyaccess. Refer to figure 2-8 for parts identification
throughout the next section.
Starting at the valves, the parts of the Fluidics
Panel are numbered counter-clockwise.
1. Valve 2 (on dual systems only)
2. Pressure Transducer 2 (on dual systems only)
3. Gas is controlled by the toggle valve. Lever ON
pressurizes the manifold.
4. Pump 2 (on dual systems only)
5. Column Outlet
6. Mixing Manifold 2 (with integrated reagent
filter) (on dual systems only)
7. Heated Reactor
filter and over-pressure relief valve)
9. Over pressure Relief valve
10. Pump 1
11. Pressure Transducer 1
12. Valve 1
13. Ambient reactor (on dual systems only)
14. Reactor outlet and union to connect to
detector
5
2
4
1
6
13
11
12
10
14
Figure 2-8

2.8
Section 2
O verview
Quick Change Reactor

The standard reactor is a PTFE capillary
tube 0.011 I.D. wrapped on a heated
mandrel. The narrow diameter reduces
band-spreading, and the PTFE is corrosion
resistant. The reactor can be easily switched
if it becomes blocked or if a different
volume is required. The electronics of the
reactor are contained within the Pinnacle PCX, making
replacement affordable and easy.
Figure 2-9
Volumes ranging from 0.15 ml to 3 ml are available. The most commonly used volumes are available off the
shelf, but if you require a volume that is not in our price list, we can make it for you.
There is a 500 psi relief valve in case there is a blockage in the reactor or detector (Item 9 on Fluids Panel).
from Detector Exit
There is a 100 psi (5 bar) back-pressure regulator

(cat. # 3102-9025) on the exit line from the detector; it
suppresses boiling inside the hot reactor and prevents
bubbles from forming in the detector flow cell. This in turn
provides for a smooth baseline and therefore high-sensitivity.
Flow direction
to Waste
Figure 2-10

2.9
Section 2
O verview
Pump Compartment
This compartment is located on the right
side of the Pinnacle PCX. It contains the
reagent pump(s), gas manifold, and
electronics for the heated reactor and
valves(figure 2-11).
The only time you will need to access
this panel is for pump piston seal
replacement. All other times, qualified
personnel should make any repairs.
Gas Manifold
Pump 2
Piston
Wash
Valve
Motor
Heated
Reactor
The Pump, Valves, and Heated Reactor

are described in other sections.
Here you will see the Piston Wash feature
for the reagent pumps. The Piston wash
is a 1/4 OD tubing connected to the
back chamber of the syringe.
Pump 1
Figure 2-11
Gas Manifold
The Gas Inlet fitting is a 1/4-28 fitting
located on the back panel of the Pinnacle PCX
(reference fig 2-15). The inert gas source is
connected via a 1/8 OD Air Barrier tubing,
and the pressure is regulated to 5psi before
pressurizing the reagent reservoirs.
The gas regulator requires an input pressure
of 4575 psi (35 bar) to function properly.
The manifold has a safety relief valve that opens
at about 10 psi to prevent dangerous overpressurizing of the reagent reservoirs.
Figure 2-12

2.10
Section 2
O verview
Display Module
The display module (figure 2-13) is the
information panel of the Pinnacle PCX.
It displays:
the flow rate of each pump, the set
point and current temperatures of
the column and reactor the Pump
pressure(s), which are also indicators
of the pressure inside the post-column
system and status information.
Stop LED
Stop
Mode
Select
Figure 2-13
In addition to the electronic information on

the LCD, the display module contains 3 buttons and one LED.
STOP - this button is used for an instant stoppage of the instrument in the case of an emergency. If this button is
pushed, the instrument will cease all activity, regardless of what is happening.
The STOP LED will illuminate when the Pinnacle PCX has been stopped manually by the user. When STOP is
pressed, the LED will glow red, indicating that there has been an emergency manual stoppage. To reset the
instrument after emergency stop, turn instrument OFF and then turn it back ON.
MODE - Used during initial installation to determine the IP address. This function is for instruments that are
connected to a company network.
SELECT - Used during initial installation to determine the IP address. This function is for instruments that are
connected to a company network.

2.11
Section 2
O verview
Electronics Compartment
This compartment contains the central nervous system
of the Pinnacle PCX the main PC board. From here, all
communication is coordinated.
This compartment is separate from the pump compartment for
two reasons:
1) To segregate the liquid end form the electronic end to avoid
any damage to the PC board by reagents and liquids
2) To keep the board cool from the warm action chamber of the
pumps
This compartment also contains:
Power supply
Cooling fans
Column oven cooling mechanism
Figure 2-14
Since this compartment is very sensitive to liquids, and shocks, it is strongly recommended that no repairs are
attempted by the user.
If there is a need for the user to open this panel,
please do so only under the recommendation/
instruction of qualified Pickering support personnel.
Back Panel
This panel contains:
Communication Ports
Gas Inlet
Power
Fuses
Vents and other openings
USB Port
Gas Inlet
Ethernet Port
Relay Input
Power Switch
and Main Fuse
COMMUNICATION PORTS
USB Port
The USB port on the Pinnacle PCX connects directly to one of the USB ports on the computer.
Figure 2-15

2.12
Section 2
O verview
Relay Ports
The relays will be explained in the operations chapter under Site Requirements.
Ethernet Port
This port is used for connecting the Pinnacle PCX to a network.
POWER SWITCH AND MAIN FUSE
The power connector is a standard IEC 320 type connector. Use the appropriate power cord for your local wall
outlet and electrical code.
The 120V version comes with a standard North American cord set.
The 240V version comes with a cord set used in much of continental Europe.
Your local reseller may have provided the correct local cord set. If your local power outlets are different, you
will need to obtain the appropriate grounded cord set.
The main power switch is located in the power connector assembly.
The fuse holder is located in the power connector assembly. To change the fuse, first remove the power cord
from the connector. Carefully pry out the fuse clip with a small screwdriver. Replace with the specified-type
fuse: 2 ea, 5mm x 20 mm, 6 A, time lag.
Warning. Ensure that the power cord is disconnected before replacing a fuse. Use only the specified-type fuse.
Attention. Assurez vous que le cable secteur nest pas connect avant de changer un fusible.
Warnung. Sicherungen drfen nur bei nicht angeschlossenem Netzkabel ersetzt oder gewechselt werden.
Cuidado. Asegrese que el cable de red est desconectado antes de instalar o cambiar un fusible.
Attenzione. Assicuratevi che il cavo di alimentazione sia scollegato prima di installare o sostituire un fusible.
Waarschuwing. Zorg dat de voedingskabel losgekoppeld is, voordat een zekering wordt geplaatst of vervangen.
Avvertimento. Fare atenzione che la corda del voltaggio sia staccata prima di cambiare valvole. Usa solo
valvole di capacit precisata dalla fattoria.

2.13
Section 2
O verview
Safety Features in the Pinnacle PCX

The Pinnacle PCX system has features
designed into the instrument and operation
that will prevent reagent back-flow onto the
column and bursting of reactor tubing due to
a blockage.
Over-pressure
relief valve
Post-column reagent can immediately damage

the analytical column if the reagent flow is
diverted in the wrong direction by a lack of
HPLC flow.
Inline
This is prevented by:
Check valve
1) In-line Check Valve
A one-way check valve placed
before the column prevents the
flow of liquid in the direction of
the column.
Figure 2-16
2) Over-pressure Relief Valve

In the rare event there is a blockage in the post-column system, an integrated 500 psi over-pressure relief valve
will open and divert the liquid into the drip tray. This will prevent a bursting of tubing or fittings. By relieving
the pressure, this will give you the opportunity to correct the blockage rather than having to replace the heated
reactor.

2.14
Section 2
O verview
Standard Configurations of Pinnacle PCX

The Pinnacle PCX is available in standard configurations for one-reagent or two-reagents, standard or micro
volume, 120V or 240V operation, and is shipped completely assembled, calibrated, and tested. Custom
instruments are also available; contact Pickering Laboratories for a quote.
LCD
LCD
Valve
Ambient
Reactor
Transducer
Transducer
Valve
Check
Valve
Dual Reagent System
Pump
Pump
Figure 2-17
Single Reagent System
Figure 2-18
The one-reagent instrument consists of a single reagent pump, valve, heated reactor, column heater, backflow
and over-pressure safety devices, filters, reagent reservoir, gas manifold, Air Barrier gas tubing, and other
accessories.
The two-reagent Pinnacle PCX contains two reagent pumps, two valves, heated and ambient reactors, column
heater, backflow and over-pressure safety devices, filters , reagent reservoirs, gas manifold, Air Barrier gas
tubing, and other accessories.
The Pinnacle PCX includes one pressurized reagent reservoir for the one-reagent system and two for the tworeagent system.
The instrument also includes a 1L wash bottle and a 1L flush bottle for the piston wash and flush feature.
3.1
Section 3
I nstallation
Section 3
Installation
3.2
3.4
3.5
3.6
3.6
3.8
3.10
3.10
3.11
Site Requirements
Instrument Unpacking and Preparation
Computer Connections
Software Installation
Pump and Autosampler Connections
Eluant Priming
Column and Guard Installation
3.12 Reservoir Connections
3.13 Reagent Pump Preparation
3.14 Running a Chromatogram
3.15 Shutdown
Installation Checklists (see Appendix)
The Pinnacle PCX instrument is shipped in one carton. Application Kits may be shipped in one or more cartons
each. Report any carton damage to the carrier. Unpack all cartons and review the contents using the Packing
List to ensure that your order is complete. If any items are missing, immediately contact Pickering Laboratories
at (650) 694-6700 or by fax at (650) 968-0749.
Store any standards in the freezer or refrigerate immediately upon arrival. Pickering Amino Acid, Carbamate,
and Glyphosate columns are shipped with test mixtures. Remove the vials from the box and freeze upon arrival.
While installing the Pinnacle PCX complete the Installation Checklist located in the Appendix. When the
installation is completed, fax or mail the checklist back to Pickering Laboratories and place in the front of the
Operation Manual under the tab labeled Installation. If the customer requires it, complete the IQ/OQ procedure
(also located in Appendix) and add that to the tab labeled Installation.
Read all installation instructions and material safety data sheets (MSDSs) before operating your post-column
derivatization instrument and HPLC system.

3.2
Section 3
I nstallation
Note: Before the Pinnacle PCX can be installed and qualified properly, the HPLC must be completely installed
and in good working order (including pump, injector, detector and data collection system). The user prior
to Pinnacle PCX installation must remove all organic compounds that are immiscible with the Pickering
Laboratories eluants as well as any hazardous chemicals.
The Pinnacle PCX uses three main styles of fittings.
1. Lite-touch fittings
These are 10-32 nut with 2-part ferrules for 1/16" OD tubing. These fittings are found at the column
connections, and at the inlet to the reagent filters. (nut: 1452-0118, ferrule: 1452-0117)
2. Long PEEK fittings
These are 1/4-28 nuts with 2-part ferrules for 1/8" OD tubing. These fittings are found at the inlet and outlet of
the reagent pumps. Low-pressure gas and reagent fittings are 1/4-28 x 1/8 inch size. (nut: 1452-0116, ferrule:
1452-0115)
3. Short PEEK fittings
These are 1/4-28 nuts with 2-part ferrules for 1/16" OD tubing or 1/8" OD tubing. These fittings are found at
the connections to the electronic valves and transducers. (nut: 1452-0113, ferrule: 1452-0115)
Pickering Laboratories supplies all the matching nuts and ferrules needed for normal installation.
Site Requirements
PINNACLE PCX SITE REQUIREMENTS
The minimum bench top space required for the Pinnacle PCX system is approximately 32H x 16W x 20D inches
(81 x 41 x 51 cm), both doors fully opened, with bottles and electrical connections in place. The Pinnacle
PCX weighs approximately 67 lbs (30kg) for simplex systems, and approximately 77 lbs (35kg) for duplex
systems. The minimum bench space does not include the HPLC system. The total space requirement depends
on the brand and model of HPLC.
For most cases, it is best to place the LC pump and injector system on the left side of the Pinnacle PCX, and the
detector on the right.
In addition to the power outlets required for the HPLC system, one grounded outlet will be needed.
Nitrogen is required to pressurize the reagent reservoir(s). The Pinnacle PCX requires gas pressure of
45-75 psi (3-5 bar) at the gas inlet. An adaptor from the gas regulator to 1/8 inch OD tubing is required.
To minimize oxidation of the TRIONE ninhydrin or OPA reagent, use oxygen-impermeable tubing for the entire
gas supply line (Air Barrier or metal).

3.3
Section 3
I nstallation
Note: If TRIONE is to be used for Reagent 1, Nitrogen must be used to prevent out-gassing.
A waste container should be provided for the waste lines from the Pinnacle PCX and the HPLC detector.
HPLC SYSTEM REQUIREMENTS
Since every HPLC is different, the following procedure has been generalized. Before attempting to connect any
tubing, examine the HPLC setup, and determine the best possible means of making the connections. Small ID
tubing (0.011") should be used wherever the sample is in the flow path.
Important! If the system will be used for amino acids, glufosinate, glyphosate, polyamines, or diquat &
paraquat analysis, be aware that the column regenerant is strongly alkaline. Any polymers or other materials in
the HPLC pump, injector, needle seat, and detector must be compatible. For example, the standard rotor seal in
Rheodyne injector valves is Vespel polyimide, which is not recommended at pH >9; a Tefzel or PEEK rotor
seal must be installed.
For all applications, the pressure rating of the detector flow cell must be > 110 psi (7.5 bar)
FOR AMINO ACID ANALYSIS
Pump
Minimum ternary gradient elution
Piston wash capability is preferable
Injector
Tefzel or PEEK rotor seal for injector valve
Tefzel or PEEK needle seat if it is an autosampler
FOR GLYPHOSATE ANALYSIS
Pump
Minimum binary gradient elution
Piston wash capability is preferable
Injector
Tefzel or PEEK rotor seal for injector valve
Tefzel or PEEK needle seat if it is an autosampler
For water samples, at least 200 l injection

3.4
Section 3
I nstallation
FOR CARBAMATE ANALYSIS

Pump
Minimum binary gradient elution
Injector
For water samples, at least 200l injection
For all other applications, review the method notes for chemistry requirements.
HPLC RELAY REQUIREMENTS
For HPLC systems other than Agilent 1100, or 1200, the Software and system must be capable of sending a relay
signal to an external piece of equipment to achieve synchronization.
Chemstation version 9.0 or higher is needed for Agilent 1100 or Agilent 1200. Pinnacle PCX software will
communicate with Chemstation directly no relay connection is needed.
Any machine that drives this relay input shall provide a relay contact pair that is electrically isolated from all
other electrical devices. The relay signal must have:
Relay detection voltage

24 +/- 2 V
Relay detection current
Approximately 1 mA
COMPUTER REQUIREMENTS
For the Installation, we strongly recommend installing the USB cable.

Use of the network connection is optional at the users discretion.
The computer must have:
Microsoft Windows XP, Vista, and Windows 7.
Minimum of one extra USB port for installation
Available Memory: Minimum 10Mb
Instrument Unpacking and Preparation

UNPACKING
Unpack all cartons and review the contents using the Packing List to ensure that all of the items are present. If
any items are missing, immediately contact Pickering Laboratories, Inc.

3.5
Section 3
I nstallation
Toll Free: (800) 654-3330

International: (650) 694-6700
Email: support@pickeringlabs.com
Internet: www.pickeringlabs.com
Unpack the instrument and place it on the bench. Place it so there is enough clearance between the Pinnacle
PCX and the HPLC, detector, and the edge of the bench.
Ensure that there is enough room to open the column oven door and the fluidics door.
Ensure that there is at least 3 inches clearance between the outlet vent at the back of the Pinnacle PCX and any
walls or other instruments. This is very important for proper cooling of the column oven.
PREPARATION AND INSPECTION
Place the Pinnacle PCX on the bench.

Examine the external chassis for damage.
Open the column oven door. Check that it is straight. Check that the latch is tight and undamaged.
Open the Fluidics chamber door. Check that it is straight.
Examine the connections on the face of the fluidics panel. Check that there are no broken, bent, or loose
tubings.
Remove the right side panel. Check that the pump/s is/are firmly attached to the chassis. Uncoil the 1/4" C-flex
tubing. This tube is for the piston wash. Place the tubing on top of the instrument in the Reservoir tray.
Check that the piston wash tubing is firmly connected to the Y fitting, see figure 2-11.
Replace the right side panel.
Connect the power cord to the outlet in the back of the Pinnacle PCX, just above the power switch.

Connect the Inlet Gas line to the back of the Pinnacle PCX.
[Set the regulator on the inert gas supply to between 45-75 psi] Using either 1/8" OD Air Barrier tubing, or
1/8" copper tubing, connect the gas supply to the Gas Inlet fitting on the back panel of the Pinnacle PCX with
the 1/4-28-1/8" nuts and ferrules (PN 1452-0181 and 1452-0180).

3.6
Section 3
I nstallation
Do not connect the 1/8" Air Barrier tubings to the bottles yet. Lay them carefully in the Reservoir tray. The gas
connections will be completed later in the installation.
Carefully turn on the main gas supply. Switch the toggle valve to the ON position to start gas flow. Let the gas
system purge for about one minute. Check for leaks at the Gas Inlet connection, and check that there is gas
flowing out of the 1/8" tubes.
Switch the toggle valve OFF.
Slide the Pinnacle PCX into place on the bench. Allow a minimum of 3 inches of space for venting at the back of
the instrument.
Computer Connections
RELAY CONNECTIONS
Relay Input Connector can be used to trigger Pinnacle PCX operations.
Connect the relay cable to the Relay Input Connector on the back of the Pinnacle PCX. The opposite end of the
cable will be connected to the HPLC systems location for relay output. Refer to the HPLC operation manual for
further information.
POWER-UP
Turn on the power switch, but do not operate the buttons.
Upon power-up, the valves will find their home positions. Ensure that this happens.
Check that the LCD displays the status of the instrument.
Software Installation
Insert the CD into the computer and follow the on-screen instructions. The Installation wizard will guide you
through the installation and configuration process.
MULTI INSTRUMENT SUPPORT
Select the number of Pinnacle PCXs that will be connected to the same PC. For each Pinnacle an icon will be
created on the desktop. Configure each instrument separately.
COMMUNICATION METHOD
Select Network or USB cable. These settings can be modified later in the Configuration window of the Pinnacle
PCX software.

3.7
Section 3
I nstallation
SECURITY LEVEL
Select who can access the Pinnacle PCX program. Everyone will allow access to many users while Just me
will only one user.
REGISTER
Complete the registration form and e-mail directly to support @pickeringlabs.com or print and fax to
(650) 968 0749.
OPTIONAL
Connecting the Pinnacle PCX to the network
It is at the users discretion if they would like their Pinnacle PCX placed on their company network. This will
involve working closely with the Companys IT personnel, to determine proper address and their network
security procedures.
A network cable is provided with the Pinnacle PCX

Connector RJ45
Connection Speed 10/100-Base-T
Ethernet protocol TCP/IP
IP address User set or DHCP
DHCP IP address is assigned by a DHCP server. The network administrator must configure the DHCP server so
that the same address is reserved for and always given to Pinnacle PCX.
User set or Static IP address is set in the Pinnacle PCX. The network administrator must provide an address
specifically for Pinnacle PCX.
Note: We recommend connection to a Network Switch or Router, or direct connection to a dedicated Network
card in the PC.
Setting up or Changing User set (Static) IP address Follow the chart below:
Note: Use the MODE button to go through options and the SELECT button to select the option. Messages appear
at the bottom of the main LCD screen.
Turn Pinnacle PCX ON. While instrument is initializing press the STOP button and hold till the message reads:
BOOT: 1-04 CPLD: 2.5
DHCP IP: 192.168.111.111

3.8
Section 3
I nstallation
Press MODE button. The message will read:

RUN MAIN F/W
DHCP IP: 192.168.111.111
Follow the chart below to make changes in IP address.
Main LCD Display
DHCP IP: 152.168.111.111
Select
Mode
RUN MAIN F/W

DHCP IP: 192.168.111.111
Select
NETWORK
IP FROM DHCP
NETWORK
STATIC IP
Mode
Mode
STATIC IP:192.168.111.111
MAC:00:50:C2:44:30:45
Select
DHCP IP: 192.168.111.111

MAC: 00:50:C2:44:30:45
Mode
EDIT IP: 192.168.111.111

Select changes number
Mode moves next to position
Mode
PRESSURE UNITS
PSI
Select
PRESSURE UNITS
BAR
Restart the instrument after completing the steps above.
Pump and Autosampler Connections

Note: Do not fit the analytical column and guard yet.
Replace any mobile phase that is more than 2 weeks old, especially DI water. Rinse the reservoir bottles
thoroughly with soap and water. Wipe down the dip tubes on the caps with methanol and a clean, lint-free
cellulose tissue. Avoid touching the tubing or the interior of the reservoir with your skin and do not leave caps
and lines dangling without a reservoir because this can cause contamination.
Fill reservoirs with 80/20 Water/Methanol
Open the prime-purge valve on the HPLC. Purge each line in the system with a water/methanol mixture to flush
the system. Set the flow rate to the maximum and purge at least 25 ml through each line.
3.9
Section 3
I nstallation
pulse dampener
outlet check valve

Tee
purge valve
Plug
from solvent
switching valve
inlet check valve
To autosampler
Figure 3-1
Drop the flow rate to 1 ml/min and close the prime-purge valve to flush the lines to the injector.
If this is an amino acid system, it is highly recommended that you dead-head the pulse dampener of the pump.
This will provide reliable gradient formation, and will prevent corrosion of the low-grade steel used in most
pulse dampeners.
Use one of the two methods below for making the connections. Amino Acid systems must be connected
according to method 2.
1.Connect the outlet of the injector to the inlet of the pre-column check valve assembly.
OR
2.Connect a tee to the inlet of the pulse dampener. Connect a line from the outlet of the pulse dampener to a tee
and place a high-pressure plug in the outlet. Connect the inlet and outlet of the pump through the tee (figure
3-1).
Connect the outlet of the injector to the Check Valve assembly. Use 0.011" ID tubing and a PEEK nut and ferrule.
(PNs 1452-0118, 1452-0117, 2104-0210, 1452-0226)
Place the open end of the check valve tubing into a beaker.
Flush line from injector for 5 minutes at 1 ml/min with 80/20 Water/Methanol.


3.10
Section 3
I nstallation
Eluant Priming
Before proceeding, check for and repair any leaks between the pump and the pre-column check valve. Once
you are certain there are no leaks, do not open the connections between the pump and injector.
IMPORTANT! If any application other than Carbamates is to be used, remove water/methanol and replace
with water. Flush at least 10 ml through lines and flush
Figure 3-2
injector line. This is to prevent any precipitation issues
Column outlet tubing
between Methanol and the buffers and to prevent any
organic solvents from entering the column. Pickering
Column oven door
Laboratories cation exchange columns for Amino Acids
Rubber foam seal
and Glyphosate analysis will be damaged by organic
solvents.
Fill the reservoirs with the appropriate eluants/mobile
phases and again flush at least 25 ml OF EACH ELUANT
with the purge valve in the open position.
Analytical column
Close the purge valve, and pump each eluant at 2 ml/

min for 5 minutes.
If buffers will be used as the eluant, use pH paper to
measure the pH of the solution coming through the
tubing.
Inlet end
Column and Guard Installation

Set the HPLC to 100% of the storage solution in the
column.
Install the pre-column check valve and female nut
inline before the column over door. Reference the
Flow Diagram in section 8.5 for complete parts list of
the pre-column check valve.
Start the flow rate to 0.2 ml/min.
Connect the line from the outlet of the pre-column
check valve to the inlet of the guard column.
When liquid exits the guard column, connect the
column connector/coupler provided in the packing
kit. When liquid exits the connector, connect the
Coupler/column connector
Guard column
Check valve
to Guard
Column
Blue Peek Tubing
from HPLC
PEEK Nut
(3101-0011)
Check Valve
(3106-1007)
PEEK Nut
(1452-0226)
3.11
Section 3
I nstallation
analytical column. Connect the Column Outlet tubing (pre-formed and cut to the right length at Pickering).
Hang the column in the column oven, use care to ensure the tubing passes through the guide hooks at the top of
the column oven. The guard column should be at the bottom, and the exit of the column at the top.
NOTE: If the application is one that uses sodium or lithium buffers, use deionized water to clean any stainless
steel fittings that have come into contact with buffer to prevent corrosion.
Monitor the pressures and stop the HPLC pump when the pressure stabilizes.
from Detector Exit
Connect a 1/16 inch x 0.020" ID tubing from the outlet of the Detector
to the external 100psi back-pressure regulator (PN 3102-9025) using a
1/4-28 nut with a 1/16 inch reversed-ferrule. There is an arrow on the back
pressure regulator indicating direction of flow. Insure that the arrow is
pointing away from the detector and toward the waste line.
Flow direction
Connect the 0.020" ID PTFE tubing provided in the packing kit (PN 21010225) to the outlet of the external 100 psi back-pressure regulator. Place
the other end in an appropriately labeled waste container.
Connect the PEEK union to the outlet of the Ambient reactor (or Heated
reactor if it is a single-pump system) using a Lite-touch Nut and Ferrule.
to Waste
Connect a 0.011" ID tubing from the outlet of the union at the exit of the
fluidics panel on the right-hand side of the instrument to the inlet of the
detector flow cell.
Figure 3-3
Set the time constant on the detector to 24 seconds.

The pressure rating of the detector flowcell must be >110 psi (7.5 bar). If your detector flowcell is rated lower,
consult Pickering Laboratories.
Special Note to Hewlett-Packard 1046A end-users: Replace the 0.12 mm ID inlet tubing (red) and heatexchanger from the left side of the detector to the flowcell (behind the front panel of the detector) with a 0.25
mm ID (blue) tubing (HP Cat. No. 79881-67302) to reduce the back-pressure.
Caution! The 100psi back-pressure regulator is directional. Do not reverse flow!

3.12
Section 3
I nstallation
Reservoir Connections
Wash all of the Pickering Pinnacle PCX reservoirs with laboratory detergent and hot water.
Rinse with methanol then with deionized water.
Wipe down the dip tubes on the caps with methanol and a clean, lint-free cellulose tissue. Avoid touching
the tubing or the interior of the reservoir with your skin and do not leave caps and lines dangling without a
reservoir because this can cause contamination.
There are three types of Reservoir/Cap assemblies used on the Pinnacle PCX:
Reagent Reservoirs are 1L, 45-430, safety-coated. These are shipped with blue caps with white valve assembly.
These can be pressurized.
Wash Reservoir are 1L, 38-430, clear glass. These are shipped with blue caps with 1ea 1/4" opening and 2ea
1/8" openings. These bottles cannot be pressurized, and do not contain threads for tubing connections.
Flush Reservoir, is 1L, 38-430, clear glass, from Wheaton. These are also shipped with caps, but with 3ea 1/8"
openings. These bottles cannot be pressurized, and do not contain threads for tubing connections.
The two types of caps cannot be interchanged because the necks of the bottles are different sizes.
REAGENT RESERVOIR CONNECTIONS
Connect 1/8" Air Barrier lines from Gas Supply OUT port on the back of the Pinnacle PCX to the CHECK
VALVE on the top of the reagent cap. Use a 1/4-28 Nut and ferrule (PN 3101-0005 and 3101-0006). Dont over
tighten!
Connect 1/8" Air Barrier lines from the Reagent ports of the valves (No 3) ports at the front of the Pinnacle
PCX to the empty port on the top of the reagent cap. Use a 1/4-28 Nut and ferrule (PN 3101-0005 and
3101-0006). Dont over tighten!
Put the cap on the bottle.
Toggle the Gas switch to ON and check that gas is flowing through the reagent cap. Close the stopcock under
the vent hole to pressurize the bottle. Open the stopcock under the reagent line before refilling the pump.
Caution! The reagent bottles are specially coated with a protective polymer to ensure operator safety if the
reservoirs should become over-pressurized. Non-coated bottles must not be substituted in the Pinnacle PCX
system. Replacement 1 L, 2 L, or 5 L reagent bottles may be ordered directly from Pickering Laboratories.

3.13
Section 3
I nstallation
Reservoirs should be labeled with an appropriate label using the GLP of the laboratory.
Fill the Flush reservoir with water or 80-20 water/alcohol (IPA or Methanol)
Fill the Wash reservoir with 90-10 water/alcohol (IPA or Methanol).
Change Wash and Flush solutions at least once a week to prevent contamination.
Rinse the Reagent reservoir(s) with a small amount of reagent or diluent, and then if necessary prepare the
post-column reagents as described in the appropriate Application section of this manual.
FLUSH RESERVOIR CONNECTIONS
Take the 1/8" FEP tubes leading from the Flush (No. 5) position on the valves and slide the tubing through the
1/8" openings on the proper cap. This will be a snug fit to prevent slipping.
(PISTON) WASH RESERVOIR CONNECTIONS
Take the 1/4" C-flex tubing leading from the piston wash of the reagent pump(s) and slide it through the 1/4"
opening in the proper cap. This will be a snug fit to prevent slipping.
Reagent Pump Preparation

All of the reservoirs should now be filled with the appropriate solutions.
Ensure that the Flush and Piston Wash lines are immersed in liquid.
The Pinnacle PCX has a maximum of two waste lines that must be fed into a waste container. They can either be
fed into a separate container, or into the waste stream of the HPLC. These are the 1/8" OD clear FEP tubes that
are connected to the Waste port (No. 6) on the valves.
Dual pump systems will have two lines, and single pump systems will have one.
In the Pinnacle PCX software, select Empty pump(s) under the Control menu. Select Both pumps for
2-pump system. The valve(s) will move to waste (1-6) position and pump(s) will dispense all the liquid.
In the Pinnacle PCX software, select Flush pump(s) under the Control menu. Select Both pumps for
2-pump system. The system will flush pumps using Flush solution. The pump(s) will be empty at the end of
Flush cycle.

3.14
Section 3
I nstallation
Running a Chromatogram
Look in the column box for the gradient program and conditions.
Set the HPLC pump to run the starting eluant conditions, and while the system is equilibrating, set up the
gradient method in the HPLC software. We recommend that equilibration is set up at the end of each run (as a
post-run, if possible). Equilibration time should be at least 5 min long to give Pinnacle PCX enough time to refill
between the runs. If using relays to synchronize with Pinnacle PCX set up relay signal at time 0.0 in the HPLC
method. Contact your HPLC support representative if you have questions about setting up relay signal.
Create a sequence for the HPLC system.
Set up method and sequence for Pinnacle PCX (refer to section 4 OPERATION of this manual). Load the
sequence. Make sure correct sequence name and method name are displayed in the status bar at the bottom of
the Pinnacle software (If sequence and method fail to load: close Pinnacle PCX software, reopen Pinnacle PCX
software, reload sequence and method).
In the Pinnacle PCX software, select Enable in the Control menu. Column and reactor heater will be turned
ON. Wait for temperatures to reach the set point.
Select Refill pump(s) in the Control menu. Select Both pumps for 2-pump system. The valve(s) will move
to reagent (3-4) position and the pump(s) will refill enough reagent for one run.
NOTE: Pinnacle software will calculate needed reagent volume based on your run time and reagent flow rate.
Some extra volume will be added to that to ensure pumps will not run out of reagent during operation.
Select Pump(s) ON in the Control menu. Select Both pump(s) for 2-pump system. The valve(s) will move
to reactor (1-2) position and pump(s) will start dispensing reagent at set flow rate. Wait until the pressures are
stable.
Select Sequence Start Sequence. Run time (0.0) will be displayed in the Status Bar at the bottom of the
Pinnacle PCX software. The reagent pump(s) will start dispensing reagent. Pinnacle now waits for injection
signal from HPLC in order to start running the method.
For TRIONE Amino Acid Analysis:
Set the detector wavelength(s) to 570 and 440nm (if applicable).

3.15
Section 3
I nstallation
Do four runs of the Amino Acid Standard that was provided with the chemical kit. Inject 10l.
Discard the first injection.
Compare the chromatogram with that of the QC test of the system and column.
Verify that the system is functioning by using the IQ/OQ documents as reference (See Operation Manual,
Appendix)
For Carbamate and Glyphosate Analyses:
Set the detector excitation wavelength to 330 nm and the emission wavelength to 465 nm
Do at least four runs of the appropriate test mixture. Inject 10ul.
Discard the results of the first injection.
Compare the chromatogram with that of the QC test of the system and column.
Verify that the system is functioning by using the IQ/OQ documents as reference (See Operation Manual,
Appendix)
Shutdown
Upon completion of the analyses, use one of the following two procedures to shut down the Pinnacle PCX
system properly. These procedures can prevent potential column damage, reaction coil blockage, high
background fluorescence, reagent precipitation, or other problems.
The Pinnacle PCX must be flushed out at the end of a series of injections, and the reactors must be cooled
down. If the instrument is simply stopped, with reagent inside the reactor in a hot state, with no movement, then
the heated reactor will become blocked. It is very important for a long useful life that the reactors be flushed
out until the temperature is cool.
MANUAL SHUTDOWN
You may shutdown the Pinnacle PCX manually by pressing/selecting the Disable function in the Control menu
of the PCX control software.
If you choose this function, allow the HPLC to pump for at least 30 minutes to allow for the reactor to cool and
to flush reagent from the reactor.
OR
AUTOMATIC SHUTDOWN
Create a Shutdown Method for Pinnacle PCX according to section 4 of this manual. Set it up as the last
method in the Pinnacle PCX sequence. Make sure pump flow rate is set to 0 mL/min and reactor temperature
is set close to room temperature. You can leave the column at the operating temperature or set it at room
temperature.
Create corresponding slowdown Method for HPLC. Set it up as the last method in the HPLC sequence. Make
sure HPLC flushes column and reactor with column storage eluant for at least 30 min.

3.16
Section 3
I nstallation
RECOMMENDED ACCOMPANYING HPLC SLOWDOWN METHOD

Set the HPLC to 100% Storage Eluant (see application section for proper eluant for your column), and set the
HPLC pump at the normal analytical flow rate. Choose an eluant that elutes contaminants from the column; for
example, methanol for a reversed-phase column and regenerant for an ion-exchange column.
Time (min)
% Storage Eluant
Flow (ml/min)
100
Analytical flow rate*
30
100
30.1
100
0.0 or 0.02mL/min
*Follow instructions that come with the column.

4.1
Section 4
P innacle PCX O peration
Section 4
Pinnacle
PCX Operation
4.1 Software Overview

4.2 Status Window
4.4 Menu Options/Functions
4.6 Configuring your Pinnacle PCX
4.7 Log Files
4.7 Creating and Editing Methods
4.10 Creating and Editing Sequences
4.11 Tutorials
4.14 Running Post-column Analysis Using Pinnacle PCX
4.17 Stop/Reset Instrument
4.17 Pinnacle Shutdown
Software Overview
Pinnacle PCX can be controlled only with PC using Pinnacle PCX software. The system can be connected to PC
through USB or Ethernet cable.
Pinnacle PCX software controls column and reactor heaters, electronic valves and pumps. It allows to create,
store and run post-column methods and sequences. It monitors reactor and column temperatures, pump
pressures and flow rates. It keeps logs that records system parameters and HPLC flags.
COMPUTER REQUIREMENTS
Microsoft Windows XP, Vista, or Windows 7 operating environment
Minimum one free USB port or network connection
Available memory: 10 Mb
HPLC SYNCHRONIZATION
Pinnacle PCX needs to receive injection signal from HPLC to synchronize operation.
For HPLC systems other then Agilent 1100 and Agilent 1200 the HPLC software and system must be capable of
sending a relay signal to an external piece of equipment to achieve synchronization.
Agilent Chemstation version 9.0 or higher is needed for Agilent 1100 or Agilent 1200. Pinnacle PCX software will
read injection signal from Chemstation directly no relay connection is needed. If any other software program
is used to control Agilent 1100 or Agilent 1200, relays board should be installed on the Agilent pump.

4.2
Section 4
Any machine that drives the relay output shall provide a relay contact pair that is electrically isolated from all
other electrical devices. The relay signal must have:
Relay detection voltage
Relay detection current
24 2V
Approximately 1 mA
Status Window
The Status window is the main screen of the Pinnacle PCX software (Figure 4-1). The Status window can be
moved, or minimized into an icon at the bottom of the PC screen. It has a menu bar on the top, status bar at the
bottom and icons for the Pinnacle PCX components.
ICONS
Pump 1: Displays the actual volume in ml
of liquid in the syringe. The status bars will
increase/decrease with volume. The actual
volume is displayed at the bottom of the icon.
Pump 2: Displays the actual volume in ml
of liquid in the syringe. The status bars will
increase/decrease with volume. The actual
volume is displayed at the bottom of the icon.
Column oven temperature: Displays the
Figure 4.1
Set and actual temperature in C. The actual
temperature is displayed at the bottom of the icon. The status bars on the column oven icon will fill in and
increase as the column heats. The reverse is true when the column cools. The icon shows the temperature of
the column oven to the nearest degree. The recommended maximum temperature for the column heater is
75C. A thermal safety switch limits the heater at ca. 80C.
Reactor temperature: Displays the Set and actual temperature in C. The actual temperature is displayed
at the bottom of the icon. The reactor temperature is measured to the nearest degree. The status bars on the
reactor oven icon will fill in and increase as the reactor heats. The reverse is true when the reactor cools. The
recommended maximum temperature for the heated reactor is 130C. Above this temperature the reaction coil
begins to lose strength. A thermal safety switch limits the heater at ca. 150C.
Pressure: Displays the actual pressure in bar or psi. Pump 1 pressure corresponds to the actual pressure on
Pump 1, and through post-column system beginning at the first mixing tee. Pump 2 pressure corresponds to
the actual pressure on Pump 2, and through the post-column system beginning at the second mixing tee.
Flow: Displays the actual flow rate in ml/min of the reagent pumps. When the pump is dispensing at the
analytical flow rate, the black square in the icon will move slowly in a clockwise direction. When the pump is
refilling, the icon changes to an alternating segments that rotate in a counter-clockwise direction.

4.3
Section 4
MESSAGE AREA
The message area gives details about the status of the instrument. This area describes s, and actions. Some
sample messages include:
Enabled Heaters are On
Instrument Stopped
Sequence Stopped
Finishing Current Run
Sequence Done
No Relay Signal
STATUS BAR
The status bar at the bottom of the screen displays the running status (elapsed run time, refilling, ready),
loaded method, loaded sequence. The status bar gives very basic information for a quick glance for method
loaded and run time.
If in Run mode:
Run Status, i.e. 3 of 7
Elapsed run time in minutes
BUTTONS
The buttons located on the right side of the Main Window emulate the buttons on the right of the LCD on the
Pinnacle PCX instrument.
Stop: This will stop the instrument. It does not matter what task the instrument is performing when this is
pressed. The Pinnacle PCX will stop whatever it is doing and will not resume until the user has Reset the system.
The button activates a red LED when the instrument is stopped. This is an emergency function designed to
allow the user to fix any catastrophic problems before they do damage or make a mess.
Mode and Select buttons are used during installation and function only on the instrument.

4.4
Section 4
Menu Options/Functions
FILE
Import/Export exports existing methods and sequences into a file. The file then can be imported to

another instance of the Pinnacle PCX software.
Exit
closes Pinnacle PCX software.
CONTROL
Enable

Refill pump(s)

Flush pump(s)

Empty pump(s)

Pump1 ON
Pump2 ON
Both pumps ON

selecting Enable will cause the reactor and column heaters to begin heating. This will not
start the pumps. The Enable button changes to Disable when pressed. Press Disable for the
instrument to stop heating. Instrument must be enabled before turning pump(s) ON or
starting the sequence.
this option will refill pumps to the volume calculated for the currently loaded method. If no
method is loaded the full syringe will be refilled. For two-pump instrument there is a choice
of refilling pump1, pump2 or both pumps. The pump(s) must be refilled before the
sequence is started. The pump(s) will refill automatically between the runs.
this option will flush the pumps using solution from the flush bottle. For two-pump
instrument there is a choice of flushing pump1, pump2 or both pumps.
this option will empty pump(s). For two-pump instrument there is a choice of empting
pump1, pump2 or both pumps.
turns on Pump1. This option changes to Pump1 OFF when selected.
turns on Pump2. This option changes to Pump2 OFF when selected.
turns on both pumps. After this option is selected, Both Pumps OFF option becomes
available.
CONTROL menu choices are not available when sequence is running

METHOD
Edit/Delete

Print
this option opens Method editing screen where Methods can be created, edited, saved or
deleted.
this will print Method information to default printer.
SEQUENCE
Edit/Delete

Print
Load
this option opens Sequence editing screen where sequences can be created, edited, saved,
loaded or deleted.
this will print Sequence information to default printer.
this loads the selected sequence.

4.5
Section 4
Start sequence starts the loaded sequence. Instrument must be enabled and pump(s) must be refilled

before starting the sequence. After Start Sequence is pressed the following choices become

available:
Stop sequence after this button is pressed the Pinnacle PCX finishes current run then stops the pump(s)

and turns the heaters OFF.
Pause sequence this option allows the Pinnacle PCX to finish current run and then stops the pump(s). It

leaves the heaters ON. The sequence will continue after Resume Sequence is selected.
INSTRUMENT
Configuration
Maintenance

opens the configuration screen.

gives the selection of the following operations:
Flush Instrument flushes the pumps and the instrument using solution from the flush
bottle. Create and load a method for Pinnacle PCX with suitable reagent flow rates, reactor
and column temperature before executing this command. Make sure HPLC is running to
avoid backflow into the column.
Change seals prepares the pump(s) for seal change. Follow directions on the screen to
complete this procedure
Prepare for storage/shipping flushes the pumps and the instrument in preparation for
long term storage or shipping. Follow directions on the screen to complete this procedure.
Pump and valve seal pressure test & system pressure test. Builds up pressure to test
the integrity of the seals after a PM.
INSTRUMENT
menu choices are not available when sequence is running.
STOP/RESET

This option performs the same action as Stop button on the front of the instrument. This
option is used for an instant stoppage of the instrument in the case of an emergency. The
instrument will cease all the activities, regardless of what is happening. The pump(s) and
heaters will be turned OFF and all the loaded methods and sequences will be cleared. After
STOP is selected the option will change to RESET. Select RESET function when you are
ready to start running the instrument again.
HELP
Index
this will open searchable Help features for the Pinnacle PCX software.
Send Log to
Support

About
saves logs to file that can be e-mailed to support@pickeringlabs.com . Logs carry

information about instrument status from the last 3 days and are necessary to troubleshoot
software and instrument problems.
gives software version number.


4.6
Section 4
Configuring your Pinnacle PCX

Select Instrument then Configuration to open configuration window (figure 4-2)
The tabs open corresponding windows that allow you to configure the Pinnacle PCX.
Instrument Tab:
Network: Select this option if the Pinnacle
PCX will be connected to the computer
through a companys network. Enter
the network IP address in the spaces
provided.
USB Port: Select this option if the
Pinnacle PCX will be connected directly to
the computer via a USB cable.
Column Tab:
Temperature Range: This window
Figure 4.2
displays the current minimum and maximum column temperature range for the Pinnacle PCX.
Reactor Tab:
Volume, ml: This window displays the current minimum and maximum reactor temperature range for the
Pinnacle PCX. The temperature range depends on the volume of the reactor. Enter the volume of the reactor
that you are using.
Pump Tab:
Max Volume: 70 ml: This is the volume of the syringe installed on the Reagent pumps(s).
Flow Rate: 0.05 1.5 ml/min: This is the flow rate range for the installed Reagent pump(s).
Pressure in: Bar or psi: Select bar if you would like to see the pressure displayed in bars, or psi if you
would like to see the pressure displayed in pounds per square inch.
More Tab:
HPLC type: Select the brand of HPLC that you will be using with Pinnacle PCX. This will determine the type
of synchronization between Pinnacle PCX and HPLC system.
For HPLC systems other then Agilent 1100 and Agilent 1200 the HPLC software and system must be capable
of sending a relay signal to an external piece of equipment to achieve synchronization. Agilent Chemstation
version 9.0 or higher is needed for Agilent 1100 or Agilent 1200. Pinnacle PCX software will read injection
signal from Chemstation directly no relay connection is needed. If any other software program is used to
control Agilent 1100 or Agilent 1200, relays board should be installed on the Agilent pump.
If your HPLC does not have Relay capabilities, select No connection to HPLC.
If you need to connect two Pinnacle PCX to the same PC you need to select multiple instruments during the
software installation. For each instrument a separate icon will created on the desktop that will open a main
Pinnacle software window. Configure each instrument before starting operation.
4.7
Section 4
Log Files
Pinnacle PCX records detailed information about the instrument parameters in the Log files. Pump(s) flow
rates and pressures, reagent volume, column and reactor temperature, s and flags from HPLC are all stored
for 3 days before being overwritten. Log files are necessary to troubleshoot Pinnacle PCX system and software
and should always be collected after a problem is detected. To collect Log files go to Help, select Send Log to
Support and select file from the day the problem happened. Save the file with the date stamp and e-mail it to
support@pickeringlabs.com. If you are not sure about the time collect all 3 log files.
Creating and Editing Methods

To run post-column analysis using Pinnacle PCX a post-column method must be created and executed in the
Pinnacle PCX software. To create or edit a post-column method select Method then Edit/Delete. This will open
Methods editing screen (figure 4-3). To execute methods (even as a single run) they should be part of the
sequence.
Methods can be edited when they are currently running. Once edited the method should be saved and the
sequence reloaded. New methods and methods not currently running can be added/edited/deleted at any time.
Status Bar This area is located at the top of the
Method Edit screen. It displays messages regarding
the saving, loading and deleting of methods. For
example, when a method called Test 1 is saved, this
area will display Method Test 1 saved.
Method Box This area displays all of the methods
currently in memory of the Pinnacle PCX software.
Here you can Edit an existing method, Delete a
method, or Add a new method. To perform an
action on an existing method, click on the method
name.
Add Click here to add a new method. The status
bar will display Method name added.
Figure 4.3
Delete To delete a method, click once on the
method name, then click Delete. The Status bar will display Method name deleted.
PUMPS TAB Timetable for both pumps (one table for both). Pump 2 flow rate column is grayed out for
1-pump systems. If timetables are empty the initial conditions are the conditions for the whole run.
Time, min: Enter the time in minutes to the nearest tenth, but not greater than the Runtime. (For example,
0.5 minutes)
P1, ml/min Enter the desired flow rate for Pump1, between 0.05 and 1.5 ml/min. (For example, 1.3 ml/min.
This means that Pump 1 will begin pumping 1.3 ml/min at 0.5 minutes)


4.8
Section 4
P2, ml/min Enter the desired flow rate for Pump2, between 0.05 and 1.5 ml/min. (For example, 0.08 ml/min.
This means that Pump 2 will begin pumping 0.08 ml/min at 0.5 minutes). Enter 0.0 ml/min if you want to stop
pumping reagent at a set time during the run.
Add This will add a new line below the currently selected line in the table.
Insert This will insert a new line above the currently selected line in the table.
Delete This will delete the currently selected line in the table.
COLUMN TAB Timetable for the column oven. If timetables are empty the initial conditions are the conditions
for the whole run.
Time, min Enter the time in minutes to the nearest tenth, but not greater than the Runtime. (For example,
15.5 minutes)
Temp, C Enter the desired temperature, in whole numbers, in C. (For example, 53C. This means that the
column will reach 53C at 15.5 minutes)
Add This will add a new line below the currently selected line in the table.
Insert This will insert a new line above the currently selected line in the table.
Delete This will delete the currently selected line in the table.
INITIAL SETTINGS In this section, set up the initial conditions for the Pinnacle PCX. If no pump timetable or
column timetable is required, Pinnacle will use this information for analysis. The items marked with * indicate
required parameters.
*Run Time, min In this window, enter the total run time for the Pinnacle PCX method. This must be a whole
number, greater than 1
*Equilibration Time, min Enter the time between runs. The Pinnacle PCX will use this information to
determine when to begin pumping again prior to the next injection. Minimum time is 5 minutes.
*Column t, C Enter the initial column temperature in C. Enter a whole number between 30C and 75C.
Column Type Enter the column type that is used for this application. Any characters are acceptable. There is
no limit on number of characters, but 20 are visible in the window at a time.
*Reactor t, C Enter the reactor temperature for the analysis. Enter a whole number between 30C-130C for
reactor volumes 2.0ml, or between 30C-80C for reactor volumes >2.0 ml. (The reactor volume is set in
Configuration).
Reactor Volume, ml Enter the volume of the reactor used in this application.
*Pump 1 Flow rate, ml/min Enter the initial flow rate for Pump 1. Enter a number between 0.05-1.50 ml/
min, or 0
Reagent 1 Enter the name or description of Reagent 1. Any characters are acceptable. There is no limit on
number of characters, but 20 are visible in the window at a time.
*Pump 2 Flow rate, ml/min Enter the initial flow rate for Pump 2. Enter a number between 0.05-1.50 ml/
min, or 0. Put 0.0 for a single-pump system.

4.9
Section 4
Reagent 2 Enter the name or description of Reagent 2. Any characters are acceptable. There is no limit on
number of characters, but 20 are visible in the window at a time.
ADVANCED SETTINGS
This tab allows you to designate currently open method as a Flush Method (figure 4-4). This method can be
set as a last method in the Pinnacle PCX sequence in order to flush the reagent pumps and the Pinnacle PCX
reagent lines and reactor. Pinnacle PCX will use
solution in the Flush bottle to do this. Always
set Flush Method as the last method in the
sequence and make sure corresponding HPLC
method is created and set as the last method in
the HPLC sequence. Flushing is not necessary
after every sequence and only recommended
before changing applications and long time
storage or in the case of extremely aggressive
reagents.
ACTION TABS
Save This will save the current method
Save As This will save the current method
Figure 4.4
information using a different name.
Cancel This will close the window and cancel any changes. No changes will be made to the method.
Print This will print the method to the default printer. The information printed will be: print date, operator,
method name, save date, initial conditions, pump time table, column time table.
Help This will open the searchable Help features for the Pinnacle PCX software.
Close This will close the window.
IMPORTANT PRACTICAL CONSIDERATIONS
In order to start executing the method Pinnacle PCX needs to receive injection signal from HPLC. To avoid
missing the signal it is very important to match Pinnacle PCX method run time and equilibration time to that of
your HPLC method.
HPLC pump uses equilibration to return to original conditions before the next analysis. HPLC software
programs have different ways of setting up equilibration. Most often used are the following:
- as a PostRun;
- at the end of the pump gradient table;
- as additional time before the injection (set as a PreRun or as negative time in the pump gradient table).

4.10
Section 4
Even if isocratic method is used and no equilibration time is required for HPLC pump Pinnacle PCX still needs
at least 5 min of Equilibration time to refill reagent pumps and stabilize the baseline signal before the next
injection.
If equilibration for HPLC pump is set up as PostRun or PreRun match Equilibration time in the Pinnacle PCX
method to respectively PostRun or PreRun time of your HPLC method. The run time of your HPLC method
(the actual time of the analysis) should be the same as Pinnacle PCX Run time. Pinnacle PCX delivers
reagents only during the Run time set up in the Pinnacle PCX method.
If returning to initial conditions is set as part of the HPLC pump gradient table (at the end or as a negative time
step in the beginning) consider how long the actual analysis and equilibration steps are. Match analysis time to
Run time of the Pinnacle PCX and match time of the equilibration step to Equilibration time of the Pinnacle PCX.
Creating and Editing Sequences

To create or edit a post-column sequence select
Sequence then Edit/Delete. This will open Sequence
editing screen (figure 4-5).
Status Bar This area is located at the top of the
Sequence Edit screen. It displays messages regarding the
saving, creative and deleting of sequences. For example,
when a sequence called Test 1 is saved, this area will
display Sequence Test 1 saved.
Select/Edit Sequence Box This area displays all of
the sequences currently in memory of the Pinnacle PCX
software. Here you can Edit an existing sequence, Delete
a sequence, or Add a new sequence. To perform an
action on an existing sequence, click on the sequence
name.
Create Click here to add a new sequence
Delete To delete a sequence, click once on the sequence name, then click Delete.
SEQUENCE TABLE WINDOW
Method name
Number of runs
Select the method name from

the drop down menu
1-...

Figure 4.5
4.11
Section 4
Add This will add a new line below the currently selected line in the sequence.
Insert This will insert a new line above the currently selected line in the sequence.
Delete This will delete the currently selected line in the sequence.
The end of the sequence should be with a shutdown method or a flush method.
ACTION TABS
Save This will save the current sequence.
Save As This will save the current sequence information using a different name.
Load This will load the current sequence into the Pinnacle PCX software.
Cancel This will close the window and cancel any changes.
Print This will print the sequence to the default printer. The information printed will be: print date, operator,
sequence name, sequence table (with method names), save date.
Help This will open the searchable Help features for the Pinnacle PCX software.
Close This will close the editing window.
EDITING THE RUNNING SEQUENCE:
- select the Sequence name from the Sequence Box.
- change number of runs of the currently running Method or add a new line to the Sequence anywhere below
the Method in progress.
- select Save and then Load from the Actions tabs.
- check number of runs on the bottom of the main software window to confirm that changes has taken affect.
Tutorials
CREATE A METHOD
The following is a tutorial on how to create a method and start a run on the Pinnacle PCX. It is not intended to
be used for a real run because the flow rates and temperatures are not representative. If you like, you may use
this tutorial as a guide to create your own methods.
This example is a fictitious method and is not intended to analyze samples.

Double-click on the Pinnacle PCX icon on your desktop. This will open the main Status Window of the Pinnacle
PCX software.
Go to Method, then select Edit/Delete. This will open the Method Edit Screen. Here you will set the initial
conditions, flow rates, temperatures for your method.

4.12
Section 4
Click Add Method.

Type a Name for your method. For this example, type Tutorial, and click OK.
The Pinnacle PCX software has now added the method Tutorial to the list of available methods. Highlight this
method if it is not already done so.
Under Initial Settings Tab, type:
Run Time, min: 60
Equilibration Time, min: 5
Column t, C: 38
Column Type: 1154150 Na cation exchange
Reactor t, C: 36
Reactor Volume, ml: 0.5
Pump 1 Flow rate, ml/min: 0.3
Reagent 1: Hypochlorite
Pump 2 Flow rate, ml/min: 0.3
Reagent 2: OPA
Click on the Pumps Tab, then click once in the box under Time. The cursor should now be flashing here. Type in
your desired Times and flow rates below. Use the Add button to add lines to the table. Use the data from the table
below as a guide. Time is the elapsed time in minutes. P1 and P2 are the flow rates of each pump in ml/min.
Time, min
P1, ml/min
P2, ml/min
1.0
0.3
0.5
1.6
0.8
0.0
3.8
1.0
0.05
12.5
0.0
0.05
30
0.1
0.0

4.13
Section 4
Next, click on the Column Tab, then click once in the box under Time. The cursor should now be flashing
here. Type in your desired Times and temperatures below. Use the Add button to add lines to the table. Use
the data from the table below as a guide. Time is the elapsed time in minutes. Temperature is in C and is the
desired temperature of the column at the time specified.
Time, min
Temp, C
5.0
45
9.5
48
25
56
50
75
Now Click Save. This has saved the conditions you have created in the method Tutorial.
Click Close to close the Edit Method Window.
Next, we will go through the Create Sequence Tutorial.
CREATE A SEQUENCE
As with the above method, this example sequence is not designed to be used for analysis. It will guide you
through the steps and you may use your own method for the Sequence Table.
From the main Status Window, go to Sequence, then select Edit/Delete.

This will open the Sequence Edit Screen. Here you will set the method names, and the number of injections for
each method.
Click Create Sequence.
Type a Name for your Sequence. For this example, type Tutorial 1 and click OK.
The Pinnacle PCX software has now added the Sequence Tutorial 1 to the list of available sequences. Highlight
this sequence if it is not already done so.
Under Method Name, there is a drop down menu. Choose the Method Tutorial.

4.14
Section 4
Next, under the Runs, type in the number of injections you plan to make using this method. In this example, it
will be 3.
Your table should now look like:
Method Name
Runs
Tutorial
The number of Runs should match the number of runs you plan to make on the HPLC using that method. In this
example, your system will perform 3 injections using the Pinnacle PCX method Tutorial. The sequence table
on your HPLC system should also have 3 injections programmed.
Now Click Save. This has saved the conditions you have created in the sequence Tutorial 1.
Click Close to close the Edit Sequence Window.
Running post-column analysis using Pinnacle PCX

To start running post-column analysis using Pinnacle PCX follow the steps below:
Create analytical method(s) for your HPLC system using gradient conditions and detector settings from
Pickering Laboratories application notes or other sources. For any other HPLC software except Agilent
Chemstation, set the relay start signal close to time 0. Depending on the software the relay stop signal
can be either a few minutes after injection or at the end of the run. Contact your HPLC support representative if
you have any questions about setting up relay signal in the HPLC method.
Create post-column method(s) for your
application using post-column conditions from
Pickering Laboratories application notes or
other sources.
Set up HPLC sequence. Contact your HPLC
support representative if you have any questions
about creating and running HPLC method or
sequence.
Start HPLC pump. Increase the flow rate
gradually to avoid overpressuring the analytical column.

Figure 4.6
4.15
Section 4
Create sequence for Pinnacle PCX using your post-column method(s). Set up the same number of runs as in the
HPLC sequence.
Select Save to save Pinnacle PCX sequence.
Select Load to load Pinnacle PCX sequence. Sequence name and the first method name will appear in the status
bar at the bottom of the Pinnacle PCX software.
Select Control Enable to turn column and reactor heaters ON. Set and actual temperatures will be
displayed on the heaters icons. Wait until the actual temperature reaches the set point.
Select Control Flush both pumps if this is the first run after the installation or if the instrument was not in
use for a long time.
Select Control Refill Pump(s) to fill pumps with reagent. Empty pumps first if there is any old reagent left
or if instrument was turned OFF earlier. The pump icon should show partially filled syringe and actual volume
of reagent inside (Figure 4-6).
NOTE: If the instrument was turned OFF with partially filled pumps the icons will show alternated black and
white segments and no volume data will be displayed. This means that the instrument does not know the
position of the piston inside the syringe. Select Control Empty Pump(s). This will allow Pinnacle PCX to
find empty position.
Once reactor and column temperatures are stable and analytical column is equilibrated select Control Both
pumps ON (or Pump ON for single-pump instrument) to start pumping reagents. Wait until the pressures are
stable.
NOTE: Pinnacle PCX software will calculate needed reagent volume based on your run time and reagent flow
rate. Some extra volume will be added to that to ensure pumps will not run out of reagent during operation. Do
not run pumps for more then 5 min before starting the sequence to avoid running out during the analysis.
Select Sequence Start Sequence. Run time (0.0) will be displayed in the Status Bar at the bottom of the
Pinnacle PCX software. Pinnacle now waits for injection signal from HPLC in order to start running the method.
Start HPLC sequence. Once an injection is made Pinnacle PCX software will receive an injection signal via relays
or directly from Agilent Chemstation. After that Pinnacle will start the post-column method. Check the timer at
the bottom of the Pinnacle PCX software to make sure it matches the elapsed time of the HPLC run.

4.16
Section 4
NOTE: after starting a new sequence always make sure that first injection signal was received normally by
checking that Pinnacle PCX timer matches elapsed time of the HPLC run. Pinnacle PCX will stop the sequence if
no injection signal is received from HPLC in 15 min from selecting Start Sequence.
After the post-column method Run time is done Pinnacle PCX turns the pump(s) OFF , checks temperature and
flow rate settings for the next run and automatically refills the reagent. Pinnacle PCX will turn the pump(s) ON
5 min before the end of the Equilibration time.
Once next injection signal is received Pinnacle PCX starts the second method in the sequence. The number
of the current run and total number of runs in the Sequence are shown on the bottom of the Pinnacle PCX
software.
After Sequence is complete Pinnacle PCX turns OFF the pump(s). Message Sequence done. (Runs n of N) is
displayed in the software window.
Important Practical Considerations
Always match number of runs in HPLC and Pinnacle PCX sequences. If more samples are added to HPLC
sequence or samples are removed edit Pinnacle PCX sequence accordingly. Total number of samples displayed
by HPLC software often does not include calibrators and controls. Make sure you count total number of runs in
HPLC sequence not just number of samples before creating Pinnacle PCX Sequence.
To edit the running Sequence:

- select Sequence Edit/Delete
- select the Sequence name from the Sequence Box.
- change number of runs of the currently running Method or add a new line to the Sequence anywhere below
the Method in progress.
- select Save and then Load from the Actions tabs.
- check number of runs on the bottom of the main software window to confirm that changes has taken affect.
We recommend restarting Pinnacle PCX software after Sequence is completed.

4.17
Section 4
STOP/RESET Pinnacle PCX

In a case of an emergency there are two ways of stopping Pinnacle PCX:
On the front of the instrument press STOP button. To reset turn the instrument OFF and ON again.
In the Pinnacle PCX software select STOP. Once selected this option changes to RESET. Select RESET once the
emergency is cleared.
These option are used for an instant complete stoppage of the instrument. Pinnacle PCX will cease all the
activities, regardless of what is happening. The pump(s) and heaters will be turned OFF and all the loaded
methods and sequences will be cleared. When ready (after the problem is fixed and you have rinsed the
analytical column) load a sequence again and follow steps to start post-column analysis.
Pinnacle Shutdown
Pinnacle can be shut down at the sequence by either using a Shutdown method or a Flush method. The latter is
recommended if the instrument is be stored for a long time or if reagents will be changed.
background fluorescence, reagent precipitation, or other problems. The Pinnacle PCX must be flushed out at
the end of a series of injections, and the reactors must be cooled down. If the instrument is simply stopped,
with reagent inside the reactor in a hot state, with no movement, then the heated reactor will become blocked.
It is very important for a long useful life that the reactors be flushed out until the temperature is cool.
MANUAL SHUTDOWN

4.18
Section 4
AUTOMATIC SHUTDOWN
Create a Shutdown Method for Pinnacle PCX using the following settings as an example:
Run time:
Equilibration time:
Reactor temperature:
Column temperature:
Pump(s) flow rate:
30 min
5 min
35 C
35 C
0 mL/min
Set it up as the last method in the Pinnacle PCX sequence. Make

sure pump flow rate is set to 0 mL/min and reactor temperature
is set close to room temperature. You can leave your column at
your operating temperature or set it at room temperature.
Create corresponding slowdown Method for HPLC. Set it up as
the last method in the HPLC sequence. Make sure HPLC flushes
column and reactor with column storage eluant for at least 30 min.
Figure 4.7

Time (min)
% Storage Eluant
Flow (mL/min)
100
30
100
30.1
100
0.0

5.1
Section 5
M aintenance
Section 5
Maintenance
5.1 Maintenance Suggestions
5.2 Operating Suggestions
5.3 Proper Shutdown Procedures
5.6 Basic Maintenance Procedures
5.7 Reagent Filter Replacement
5.8 Ambient Reactor Replacement
5.8 Replacement of Heated Reactor Cartridge
5.9 Valve Maintenance
5.12 Pump Seal Replacement
5.16 Fuse Replacement
5.17 Tubing Kit Guides
Your Pickering Pinnacle PCX will require some routine maintenance to stay in top condition. Ordinarily, little
maintenance is needed beyond good operating procedures. In this section you will find some suggestions for
maintaining your Pinnacle PCX, as well as Proper Shutdown procedures for typical, Long-term, and Storage of
the Pinnacle PCX.
Maintenance Suggestions
Make copies of the blank forms in the Appendix and complete the parameter log on the photocopy.
Record the pressures for the system equilibrated under initial conditions. Keep a daily log of Column, Pump
1 and Pump 2 (if applicable) pressures for diagnostic use. Include all the settings for the pump, injector,
detector, and integrator.
Record any repairs, or problems in the Instrument Log.
Keep copies of the QC chromatograms and logs, and of the initial chromatograms generated at installation.
Create a maintenance schedule for your laboratory to ensure that the instrument is kept in good working order.
Always use PEEK nuts and ferrules in the Pinnacle PCX fluidics lines. Use of stainless steel nuts and
ferrules in the PEEK ports will void the warranty. Stainless steel nuts and ferrules will cut the threads at
the bottom of the ports, causing them to leak. If this happens, the entire item may need replacing.

5.2
Section 5
M aintenance
Operating Suggestions
Below are several helpful suggestions and bits of information which will help keep your Pinnacle PCX, columns,
and reagents in top condition. The following information is broken down into sections for convenience.
There are two items that should be part of any good laboratory practices, regardless of what equipment is being
used:
1) Always wear safety glasses or goggles, laboratory coat, gloves, and other appropriate safety-clothing.
2) Read and understand the instructions in the MSDSs shipped with the chemicals. If the MSDSs are missing,
please contact Pickering Laboratories and we can fax you a copy, or you can download them from our website
at www.pickeringlabs.com.
PINNACLE PCX
Check for leaks daily at the column fittings; the eluants can be corrosive.
Frequently observe and record the pressures and check for leaks. You may find a problem before it becomes serious.
Always use the correct fuse. Use of the incorrect fuse can seriously damage the PC board and even the valves,
reactor and pump. Use of an incorrect fuse will void the warranty.
Disconnect the power cord before removing the case of the Pinnacle PCX.
Always follow the proper shutdown procedures. See Below.
Use the proper start-up and shutdown procedures consistently (see Section 4).
Periodically Flush the instrument to ensure long life.
Do not operate the heated reactor above the boiling point of the eluant unless the back-pressure regulator is
connected to the waste line of the detector. Boiling inside the reactor can cause precipitates to block the reactor.
Operating above the boiling point without a back pressure regulator will void your warranty.
Thoroughly clean any leaks from fittings with water and dry with paper towels, especially if the solution is a
buffer or hydroxide. Standing salt and hydroxide solution are corrosive.
Soak up spills with rags, paper towels, or sponge. Clean spill-area with a wet towel and thoroughly dry. Do not
spray water directly into the instrument.
Rinse out the drip tray periodically.
MAINTENANCE SCHEDULE
Check for leaks around all fittings and record column and reagent pressures daily.
Change the reagent filter every 3 months.
Change valve and pump seals every 12 months.
Change the heated reactor and all tubing every two years.
5.3
Section 5
M aintenance
PICKERING COLUMNS
Do not operate with a column pressure above 2800 psi (193 bars) for an extended period of time. Isolate the
source of the high pressure and replace those items.
Never disconnect any fittings between the pre-column check valve and the column until the post-column system
has been shut down and depressurized (loosen the detector inlet fitting first). If the column pressure drops
below the post-column pressure, it is possible for reagent to back-flow onto the column.
When removing the column, remove the outlet fitting first.
Never pump organic solvent through an ion-exchange column. This will swell the resin and over-pressure the column.
Never pump high pH buffers or hydroxide through a silica column. This can dissolve the silica and cause
extensive damage to the post-column system by permanently blocking tubing.
When switching a system between ion-exchange and reversed-phase applications, be sure to flush the HPLC and
injector with water before connecting the column. Eluants for one analysis may damage the column for the other.
ELUANTS AND REAGENTS
Thiofluor is extremely hygroscopic. Always keep it in a tightly closed container.
Sodium borate (any grades) contains excessive amounts of heavy metal contaminants and insoluble matter.
These impurities will eventually precipitate in the reactor and flowcell. The one year warranty does not
cover damage caused by these contaminants.
If you must prepare your own borate buffer for the OPA reagent, do not use sodium tetraborate as
suggested by the EPA methods. Instead, use molar equivalents of boric acid and sodium hydroxide, because
they are available in higher purity (ACS-grade or better) and have very little insoluble matter.
Use Pickering Laboratories reagents and eluants. The quality of the chemicals is guaranteed and the cost is low
relative to the worth of your analytical results. The one year warranty does not cover damage caused by
poor-quality reagents and eluants not purchased from Pickering Laboratories.
Do not touch the interior of the mobile phase reservoirs and the dip tubes with your fingers. Amino acids in
fingerprints will cause contamination. Gloves are suggested.
Do not leave caps and lines dangling without a reservoir. To fill reservoir, transfer caps and lines into a spare
bottle or an Erlenmeyer flask filled with deionized water.
Proper Shutdown Procedures

background fluorescence, reagent precipitation, or other problems.
The Pinnacle PCX must be flushed out at the end of a series of injections, and the reactors must be cooled
down. If the instrument is simply stopped, with reagent inside the reactor in a hot state, with no movement, then
the heated reactor will become blocked. It is very important for a long useful life that the reactors be flushed
out until the temperature is cool.


5.4
Section 5
M aintenance
MANUAL SHUTDOWN
OR
AUTOMATIC SHUTDOWN
Create a Shutdown Method for Pinnacle PCX according to section 4 of this manual. Set it up as the last
method in the Pinnacle PCX sequence. Make sure pump flow rate is set to 0 mL/min and reactor temperature
is set close to room temperature. You can leave the column at the operating temperature or set it at room
temperature.
Create corresponding slowdown Method for HPLC. Set it up as the last method in the HPLC sequence. Make
sure HPLC flushes column and reactor with column storage eluant for at least 30 min.
Time (min)
% Storage Eluant
Flow (mL/min)
100
30
100
30.1
100
0.0

LONG-TERM SHUTDOWN
The above methods are used if the Pinnacle PCX will be used again within 3 days of the last run. If you plan to
not use the Pinnacle PCX for more than 3 days, we recommend that you use the Long-term Shutdown.
In this procedure, the reactor is flushed free from reagent, thus ensuring that it will be free from blockage
upon your return.
There are two ways to perform this shutdown; automatic, or manual.

5.5
Section 5
M aintenance
MANUAL LONG-TERM SHUTDOWN

Make sure your HPLC pump is running storage eluant through the analytical column (see Application section
for proper eluant) and then remove the column for storage. Replace the column with a union and the eluants
with Water and Methanol. Set the HPLC pump to 80% Water and 20% Methanol at 1.0 ml/min. This will prevent
backflow into the column during Flush procedure.
Create a Flush method as described in section 4.9 under the Advanced Settings tab of this manual. Set up a
Sequence with one run of a Flush method and load it into Pinnacle PCX software.
Select Enable function in the Control menu.
Select Instrument Maintenance- Flush Instrument. Follow directions on the screen to complete this
procedure.
After Flush Instrument is completed select Disable function in the Control menu of the Pinnacle PCX software to
turn OFF heaters and pumps.
Allow the HPLC to pump at least 30 minutes to allow for the reactor to cool.
AUTOMATIC LONG-TERM SHUTDOWN
Create a Flush method as described in section 4.9 under the Advanced Settings tab of this manual. Set it up as
the last method of your Pinnacle PCX sequence.
Create a corresponding method for your HPLC to flush analytical column with the storage eluant (see
Application section for proper eluant) and set it up as a last method of your HPLC sequence.
After sequence is completed select Disable function in the Control menu of the Pinnacle PCX software to turn
OFF heaters and pumps.
Allow the HPLC to pump at least 30 minutes to allow for the reactor to cool.
STORAGE
If the Instrument will not be used for a period longer than 2 weeks, it must be put into Storage state. This
becomes necessary because as reagents age, the chances of precipitation in the heated reactor increases.
Perform Manual or Automatic Long-Term Shutdown as described above.

Remove the reagents and replace them with a solution of 80/20 Water/Methanol.


5.6
Section 5
M aintenance
Select Refill Pump(s) from Control menu of the Pinnacle PCX software. This will flush the reagent lines from
the bottles from the remaining reagent.
Select in the Pinnacle PCX software Instrument Maintenance Prepare for Storage/Shipping. Follow
directions on the screen to complete the procedure.
Pump the storage eluant through the analytical column (see Application section for proper eluant), and remove
the column and guard.
Cap both column and guard tightly.
Replace any buffers with water and flush the HPLC lines for 5 minutes.
Replace the water with 80/20 Water/Methanol and flush the lines.
3
Place a restrictor or union in the column oven in
place of the column.
Pump 80/20 Water/Methanol through the system.
Basic Maintenance Procedures

The following section will describe the procedure
for cleaning/replacing/rebuilding key elements of
the Pinnacle PCX instrument.
For the following procedures, refer to Figure 5-1
below, which calls out the various parts of the
Pinnacle PCX.
Starting at the valves, the parts of the Fluidics
Panel are numbered counter-clockwise.
1. Valve 2 (on dual systems only)
2. Pressure Transducer 2 (on dual systems only)
3. Gas is controlled by the toggle valve. Lever ON
pressurizes the manifold.
4. Pump 2 (on dual systems only)
5. Column Outlet
filter) (on dual systems only)
7. Heated Reactor
filter and over-pressure relief valve)
5
2
4
1
6
13
11
12
10
14
Figure 5-1

5.7
Section 5
M aintenance
9. Over pressure Relief valve

10. Pump 1
11. Pressure Transducer 1
12. Valve 1
13. Ambient reactor (on dual systems only)
14. Reactor outlet and union to connect to detector
Reagent Filter(s) Replacement

Refer to Figures 5-1 and Figure 5-2 for the location of the Reagent filter(s).
There are two filters in the Duplex system, and one filter
on the Simplex system. They are located in the reagent
manifolds; Items 6 and 8.
Reagent Filter
1. Be sure that the Pinnacle PCX is shut down and

depressurized before changing a filter.
2. Remove the 1/16" fitting from the inlet of the reagent
filter.
3. Use a 7/16" wrench to remove the filter.
4. Replace the filter element. The correct filter element is
3102-9040, a 10 m frit. Tighten with fingers first, then
give it a 1/4 turn with a 7/16" wrench
Reconnect the tubings to the filter. Use care not to strip the threads, and do not overtighten.
Start the LC pump and inspect for leaks.
Figure 5-2
Note: Always use a PEEK nut and ferrule to connect to the reagent filters. They are made of soft PEEK material,
and stainless steel nuts and ferrules will cut them, causing them to leak.

5.8
Section 5
M aintenance
Ambient Reactor Replacement

The Ambient Reactor is Item 13 (on dual systems only).
1. Shutdown the post-column system and let the reactor cool for at
Figure 5-3
least 30 min.
2. Disconnect the two fingertight fittings at either end of the ambient reactor. These will be located on the
manifold (Item 6) and the inlet of the detector.
3. Loosen the screw that holds the black spindle to the fluidics panel with a 3/32" Alan wrench.
4. Remove the spindle and remove the used reactor.
5. Slide the new reactor onto the spindle and fasten to the fluidics panel.
6. Re-connect the fingertight fittings to the manifold and the union (Items 6 and 14). Do not overtighten the
fittings.
7. Start the LC pump and inspect for leaks.
Replacement of Heated Reactor Cartridge

The Heated Reactor is Item 7. Please refer to Figure 5-4 for removal of the Reactor Cartridge.
1. Shutdown the post-column system and let the reactor cool until it is a safe temperature for handling (below
30C).
2. Disconnect the fingertight fittings connected to the heated reactor. These will be located on the manifolds
(Items 6 and 8) for duplex systems or on the manifold and union in simplex systems (Items 8 and 14).
3. Loosen the retaining screw at the top of the reactor cartridge using a flathead screwdriver
(see Figure 5-4).
4. Gently slide the Reactor Cartridge out of the reactor
housing.
5. Replace the heated reactor cartridge with the proper
volume (see Appendix).
6. Reconnect the two fingertight fittings.
Do not overtighten. The inlet tubing of the
heated reactor is marked with a piece of
black shrink tube and connects to mixing
manifold I (Item 8).
Start the LC pump and inspect for leaks.
Retaining Screw

Figure 5-4
5.9
Section 5
M aintenance
Valve Maintenance
Flushing the Valves is recommended before valves replacement or resealing to remove reagents from lines and
pumps. To flush the valve follow the steps below:
Remove the reagents and replace them with a solution of 80/20 Water/Methanol.
Select Refill Pump(s) from Control menu of the Pinnacle PCX software. This will flush the reagent lines from the
bottles from the remaining reagent. Select empty pump(s) to dispense liquid from pump.
Select Flush Pump(s) from Control menu of the Pinnacle PCX software.
Now Valves are ready for maintenance.
VALVE FACE REMOVAL PROCEDURE
Replace the post-column reagents with 80/20 water/methanol.
Flush the pump connected to the valve that requires maintenance (in the software, go to Control, then Flush
Pump).
When the flush is complete, pull the Flush tubing out of the liquid to prevent siphoning of the liquid back into
the flush line.
Power down the Pinnacle PCX and turn the power back on again. This will move the valve to the blocked
position. This is important for alignment of the new or repaired valve face.
Place some paper towels under the Pinnacle PCX to
absorb any excess liquid that is in the Flush line.
Disconnect the Waste line (position 6) from the
Valve face. Hold the end of the tubing upright for a
moment to allow any liquid to drain into the waste
container.
Disconnect the remaining fittings from the valve
face.
6
Flat-head screw
Using an Alan wrench and a flat-head screwdriver,

loosen the cap screw that holds the valve indicator
plate in the center of the valve, and the 2 flat-head
screws located between ports 1-2 and 4-5.
Cap screw
5
Flat-head screw
Figure 5-5

5.10
Section 5
M aintenance
Pull the entire face horizontally forward. There will be some resistance.
If there has been any crystal build-up behind the valve, insert the jack screws
contained in the Valve Maintenance kit and tighten them until the face of the
valve comes forward and you are able to remove it (figure 5-7).
Warning: When removing the valve face with the jack screws, it is important
to remember to remove the valve using equal turns on each side.
It is important that it come off of the instrument as straight as possible to
prevent deforming the soft PEEK material.
Body
Spool
Figure 5-6
Remove the jackscrews after the valve is removed.

Inspect the face of the motor mount. Ensure that there is no excess crystal
build up. Carefully clean any crystals with a paper towel and some DI Water.
jack
scews
Using the Pinnacle Valve Seal tool, turn insert clockwise and push to remove
spool (figure 5-8).
O-RING REMOVAL
DO NOT use any sharp tools as this will scratch the soft plastic of the
insert.
Figure 5-7
For the 5 smaller O-rings (Size 4) use a length of 1/16 OD Peek tubing to
remove.
For the 2 large O-rings (Size 18) pinch o-rings with fingers and roll to remove.
Once O-rings are removed, sonicate PEEK ring and PTFE insert to remove any
crystals from valve. O-rings will be replaced.
Figure 5-8
O-RING REPLACEMENT
Clean the new O-rings first by sonicating them in a soapy water bath for 20
minutes and then rinse thoroughly with DI Water. Wear gloves and use care
not to get dust or dirt on them after cleaning.
Push the 5 new Size 4 O-rings into the ports of the PEEK ring. Port 6 (Waste
port) does not have an O-ring.
Figure 5-9

5.11
Section 5
M aintenance
Carefully slide the 2 new Size 18 O-rings onto the white center spool.
Note: Never reuse the O-rings. They take a shape after they are installed and will not conform to a new shape.
Note: Never use substitute O-rings.
Only use O-rings provided and approved by Pickering Laboratories.
INSERTING THE SPOOL INTO THE PEEK RING
Using the special Pinnacle Valve Seal tool (the same tool used to remove the
spool), carefully put the PTFE center back into place in the PEEK ring. Rotate
the insert clockwise to prevent slicing of the O-rings. Look into the valve, and
push insert toward you to ensure that no O-rings pop out.
Line up the holes in the spool so that when it is placed on the Pinnacle, the V is
between ports 2-3. This is the equivalent to the blocked position of the insert.
REPLACING THE NEW/REBUILT VALVE ON THE PINNACLE

Ensure that at the front face of the new valve, the body and spool are
completely flush. These two pieces must be level.
6
5
Figure 5-10
Slide the new valve onto the motor mount, so that the Number 1 is at the top.
Ensure that the dowels match the openings in the back of the valve.
Ensure that the V is between ports 2-3 (that is, ensure that the spool is in the home blocked position) refer to
figure 5-10.
Push the valve face firmly against the Pinnacle PCX. Ensure that the Valve Spool is firmly pushed against the
motor mount.
Replace the cap screws and tighten until you feel slight resistance. Then make 1/8 turn past snug.
Perform a pump flush to exercise the new valve and to properly position the spool within the body (remember
to put flush lines back into the solution).

5.12
Section 5
M aintenance
Pump Seal Replacement

The Pinnacle PCX uses Pickerings own pulse-free syringe pump with piston wash. The pump is specially
designed for extended seal life, however the piston seals will require periodic replacement.
The length of service to be expected from the seal depends on a wide variety of factors, including whether
proper shutdown procedures were followed, how often the system was turned on and off, and whether the
piston-wash system was wetted.
It is critically important that the seal be replaced immediately upon failure, or better yet, before failure, because
the reagent can leak into the mechanical housing of the pump and cause corrosion. When a leak occurs, you
may notice fluid on the side of the pump.
The pumps are Items 4 and 10.
SOFTWARE SET UP
To replace the seals, open the PCX control software and select Maintenance under the Instrument menu.
From the Maintenance sub-menu, select Change Seal, and follow the onscreen instructions.
Wait until the end of a run before performing this procedure to conserve reagent.
The Pinnacle will perform the Flush pump sequence, and will flush reagent out of the syringe.
Pull the Piston Wash and Flush lines out of the liquid, so they are just inside the caps. Leave enough space for
the pump to dispense any remaining liquid into the bottles.
When you are ready, click OK on the software.
The pump will retract the piston to dispense any piston wash back into the bottle.
Next, the pump will move the piston all the way forward to empty any remaining liquid into the Flush bottle.
The Valve will move to the blocked position to prevent any flow from the bottles.
Disconnect the Reagent lines from the pump head and plug the openings to prevent any spillage.
Open the panel on the right side of the Pinnacle PCX
by removing the captive screw located at the back of
the Pinnacle PCX, then push the cover back, and lift
it up.
TO REMOVE THE PUMP FROM PINNACLE PCX
Turn off Pinnacle PCX.
Disconnect the electronic pin connector from the
inside wall of the Pinnacle PCX.
Remove the two Alan bolts from the bottom of the
pump first. See Figure 5-11.
Figure 5-11
5.13
Section 5
M aintenance
Note: It is very important to remove the bottom bolts first. There are specially designed hooks in the chassis that
will support the pump.
Loosen and remove the third top screw. Use caution as this will cause the pump to lean toward you. Brace the
pump toward the wall of the Pinnacle PCX to prevent damage.
Carefully remove the pump from the chassis. If it is Pump 1, tilt the back end up and remove the pump in a
vertical position. If it is Pump 2, tilt the back end down, and remove the pump in a vertical position.
Place the pump on clean paper towels on the bench.
Wear gloves for this procedure.
REMOVE SYRINGE FROM BODY OF THE PUMP
Once the pump is on the bench, carefully remove the
syringe from the body of the pump:
Using a 3/32 Alan wrench, loosen the set screw (DO NOT
remove set screw) at the front of the syringe. It is important
to perform this step first, as it will remove the tension from
the bracket pins and cap screws (figure 5-12).
Cap Screw
Bracket
Cylinder Cap
Set Screw
Figure 5-12
Once the set screw is loose, using the 3/16 Alan wrench, loosen the two cap screws that secure the bracket to
the pump base.
Remove the bracket and cylinder cap by gently pulling the bracket ends out and away from the pins.
Carefully remove the ceramic syringe body by turning it in a
clockwise direction as you pull toward you (figure 5-13).
Warning: Never turn ceramic syringe body counter clockwise.
This will cause the Piston Head to unscrew and fall off inside of
the cylinder, making it very difficult to remove.
There will be some liquid inside the body, which will spill onto
the paper towels.
Set the syringe body aside on a clean dry surface.
The piston should be fully extended from the pump.
Figure 5-13

5.14
Section 5
M aintenance
PISTON HEAD UNIT DISSASEMBLY AND

SEAL REPLACEMENT
Note: All items to be replaced in the Pump Seal
Replacement Procedure can be found in the the
Pinnacle Pump Seal Kit (PN 1452-0122).
In the following steps, you will replace 5 items:
Brass-ended set screw
PistonHead O-Ring
Secondary Cylinder O-Ring
Piston Rod O-Ring
Three, 4 - 40 x 1/4" capnut screws and washers
Metal Piston Face

Piston Head O-Ring
Secondary Cylinder O-Ring
Piston Rod
O-Ring
Set Screw
Figure 5-14
PEEK Piston Head
O-Ring Guide
Pump Base
Using a 1/16 Alan wrench (do not use ball wrench

because it is too weak) remove the set screw from the
Piston Head (figure 5-15).
Note: Very Important! Never re-use the set screw. It has
a soft brass end that is designed to be used only once.
A new set screw is included with the seal kit. Re-use of
the set screw can cause the piston head to come loose.
Piston O-Ring
Guide
Lead Screw
Piston Head
Unit
Figure 5-15
Remove the piston head by turning it in a counter-clockwise direction. It is threaded onto the leadscrew. If
needed, use a cresent wrench to lossen the piston head.
Place the piston head on the bench with the PEEK side down. Using a Phillips Head screwdriver, remove the
metal plate by removing the 3, 4 - 40 x 1/4" capnuts and washers. These cannot be reused and should be
replaced. The piston head will disassemble into 3 parts:
1. Metal Piston Face
2. Piston Head O-ring
3. PEEK Piston Head
Blow out debris from brass-ended set screw that may be inside the piston head. Remove and discard the old
O-ring. Sonicate the metal plate, PEEK piston head, and new O-rings in a beaker of water.
Note: Do not over tighten cap screws. Over tightening will strip the threads on the PEEK piston head.
Rinse with DI water and reassemble the Piston Head using the new O-ring and cap screws. Set the reassembled
Piston Head Unit aside until Pump Reassembly.

5.15
Section 5
M aintenance
SECONDARY O-RING REPLACEMENT

To replace the secondary O-ring at the base of the pump,
first remove the piston wash connection and then back
piston by turning the lead screw 4 to 5 full turns manually.
Reverse direction of turn by 4 to 5 full turns by turning the
lead screw in the opposite direction. This will extend the
O-ring guide at the base of the pump (figure 5-16).
Note: O-ring guide will not come out with piston wash
connection still attached.
Piston Wash
Tubing
O-ring Guide
Placement of
Secondary
O-ring
Figure 5-16
Slide the O-ring guide out and replce the small piston rod
O-ring that is located at the back of the guide (figure 5-17). Slide the guide back to the base of the pump and
replace the large secondary O-ring with the new one (figures 5-14
and 5-16).
PUMP REASSEMBLY
Screw the newly assembled Piston Head Unit onto the head screw
(hand tight).
Using a new brass-ended setscrew, tighten the setscrew with the
1/16" Alan wrench. It is important to make this tight.
Figure 5-17
Lubricate the ceramic body with some methanol or isopropanol. Turning it in a clockwise direction, push it
back over the piston, using care to keep it horizontal. Ensure that the through holes are
orientated top and bottom (figure 5-18).
Place the cylinder cap onto the head of the cylinder and the
bracket on the pins, but do not tighten the cap screws. Reference
figure 5-12.
The set screw at front of cylinder has adjusment depression
to center bracket. Center the bracket by sliding it back and forth
a little bit until you feel it in the depression. Once it is centered,
tighten the set screw. Tighten the set screw so that the syringe is
tight against the base of the pump.
Figure 5-18
Finally, tighten the cap screws using the 3/16" alan wrench.

5.16
Section 5
M aintenance
REPLACE THE PUMP IN THE PINNACLE PCX

Replace the pumps in the Pinnacle PCX by following the reverse of the directions in section titled To Remove
the Pump From Pinnacle PCX on page 5.12.
Be sure that the body of the pump rests on the support brackets, and that the front of the pump is protruding
through the front of the chassis.
Note: Be very careful not to pinch any of the other wires inside this area. Be especially careful of the reactor
wires and the valve wires when removing Pump 1.
Attach the reagent and piston wash lines.
FLUSH AND REFILL PUMPS
Turn ON Pinnacle PCX.
Select Control Flush pump(s). Make sure all flush lines are immersed in solution.
After seals have been exercised for at least 60 minutes, perform Pump and Valve Seal Pressure Test followed by
a System Pressure Test. Follow the onscreen instructions.
Fuse Replacement
The fuse is on the back panel under the power switch.
Warning: The Pinnacle PCX must be shutdown to replace the fuse.
Remove the cord from the power inlet.
Use a small flat screwdriver to pry up the fuse holder then pull the fuse out.
Only use the correct type of fuse: 2 ea, 5mm x 20 mm, 5 amp, time lag
Reinstall the fuse holder and the power cord.

5.17
Section 5
M aintenance
R1
TRANSDUCER 2
R2
R23
FLUSH
1100-0500 Tubing Kit, Pinnacle, Dual Pump
R24
COLUMN
R25
W2
PUMP 2
R21
3 VALVE 2 5
4
R22
FILTER
F2
AMBIENT
REACTOR
HEATED
REACTOR
S2
TRANSDUCER 1
R14
R15
FILTER
R13
PUMP 1
R11
W1
VALVE 1 5
4
R12
F1
WASTE
TO DETECTOR
FROM
HPLC

5.18
Section 5
M aintenance
R1
FLUSH
1100-0501 Tubing Kit, Pinnacle, Single Pump
S2
COLUMN
FILTER
TRANSDUCER
HEATED
REACTOR
R14
R15
2
R13
PUMP
W1
1
R11
VALVE 1 5
4
R12
F1
WASTE
TO DETECTOR
FROM
HPLC

6.1
Section 6
A pplications
Section 6
Applications
6.2
6.3
6.4
AMINO ACIDS
CARBAMATES
GLYPHOSATE

6.2
Section 6
A pplications
AMINO ACIDS
6.2 Introduction
6.2-1 Background
6.2-2 Basic Sample Preparation

6.2-3 Reagent Preparation
6.2-4 Analytical and Post-column Conditions
6.2-5 Procedure
6.2-6 Sample Chromatograms and Gradient Programs
6.2-18 Precautions
6.2-21 Notes
Introduction
High performance liquid chromatography (HPLC) with post-column derivatization is a technique for rendering
analytes more detectable than they would otherwise be in their native forms. Post-column derivatization gives
improved sensitivity or better selectivity (reduction of interference) leading to lower detection limits. The
Pickering Laboratories Pinnacle PCX was developed to facilitate the determination of amino acids in
using sodium ion-exchange or in native samples using lithium ion-exchange columns. There are two options
for post-column detection of amino acids. The first is the use of Pickerings patented TRIONE ninhydrin
reagent, which will react with both primary and secondary amino acids. The second is the use of
o-phthalaldehyde (OPA), a fluorescent reagent that gives greater sensitivity but will detect only primary amino
acids.
A complete post-column analysis system for amino acids consists of the following components:
HPLC ternary or greater gradient pump
Manual injector or autosampler equipped with high pH compatible Tefzel or PEEK seals
Pickering Laboratories ion-exchange columns
Pickering Pinnacle PCX post-column derivatization instrument
Eluants, reagents, and standards
Visible or fluorescence detector
Chart recorder, integrator, or data system
Ion-exchange chromatography followed by post-column derivatization has been the method of choice for amino
acid analysis since S. Moore, D.H. Spackman and W.H. Stein published it in 1958work which merited a
Nobel prize.

6.2-1
Section 6
A pplications
Background
The separation is a multi-modal process wherein ion-exchange, ion-exclusion, and partition all take place.
The primary process is cation-exchange where a pH gradient mobilizes amino acids in order of their isoelectric
points; acidic amino acids such as glutamic acid elute early and basic amino acids such as lysine elute late.
Partitioning is affected by ionic strength and organic modifiers; for example threonine and serine are resolved
by partition effects. Ion-exclusion only occurs for highly acidic amino acids such as taurine.
Sodium ion-exchange is used for fast analysis of the 22 amino acids found in hydrolyzed protein or in simple
formulated products. Lithium ion-exchange is a slower technique with higher resolution to separate as many as
46 amino acids and compounds found in the complex mixtures of biological fluids or tissue extracts.
The most popular reagent for post-column detection is ninhydrin. Ninhydrin reacts with primary amines and
hydrindantin to form Ruhemanns Purple (Figure 6.2-A) which is detectable at 570nm. Ninhydrin reacts with
secondary amines to form a yellow complex detectable at 440nm. The ninhydrin reaction is carried out at
130C with a reactor volume of 500 l. The elevated temperature is required because at room temperature,
the ninhydrin reaction is very slow and takes hours to go to completion.
Figure 6.2-A

6.2-2
Section 6
A pplications
An alternative reagent system based on o-phthalaldehyde (OPA) can be used for high-sensitivity detection of
primary amino acids. OPA reacts rapidly with primary amines and Thiofluor (N,N-dimethyl-2mercaptoethylamine) under mild basic conditions to produce a strongly fluorescent isoindole derivative
(Figure 6.2-B). OPA does not react with secondary amines or aryl amines, so fails to detect Proline and other
secondary amino acids.
CH3
N
10 Amino Acid
OPA
CH3
S
O-
O
CHO
pH>9, ambient T
+
NH3
H
CHO
COOH
H
HS
CH3
Fluorescent Isoindole
CH3
Thiofluor
Figure 6.2-B
However, it is possible to detect secondary amino acids by using a two-step reaction in which they are first
oxidized and then reacted with OPA. This technique has some disadvantages, and is not often used. Contact
Pickering for details.
The Pickering Pinnacle PCX derivatization instrument for fluorescent detection of amino acids is similarly
designed to the ninhydrin instrument, except that it contains a 150 l reactor and the reacton is carried out
at 45C.
Basic Sample Preparation

The following is a general sample preparation for Physiologic Fluid and Protein Hydrolysate samples. There are
many more types of samples that can be used for amino acid analysis. For details, consult the AOAC methods
and laboratory procedures.
Native amino acids are those found free in samples such as serum, urine and other physiological fluids, plant
extracts, foods and beverages. Although preparation of these samples for amino acid analysis is much simpler
and less time-consuming than protein hydrolysis, control of pH and normality, and removal of soluble protein
are critical factors which can affect the chromatography.

6.2-3
Section 6
A pplications
The early-eluting amino acids taurine, urea, aspartic acid, threonine, serine, etc. are particularly
sensitive to pH and normality. Accordingly the samples must be held to a narrow pH range between 2.1 and
2.5, and the proper lithium ion concentration to ensure reproducibility in the early part of the chromatogram.
The later-eluting compounds are more tolerant of initial sample conditions, and their retention times are not
as likely to be affected. SERAPREP and URIPREP replace commonly-used protein precipitation reagents such
as acetonitrile, perchloric acid and picric acid, and eliminate the need for dialysis, ultrafiltration, and repeated
centrifugation steps, followed by pH adjustment.
Filter all samples through a 0.45m membrane filter. Some samples may require even more stringent
filtration, especially if colloids are present.
Samples must always be properly buffered. The ideal pH for sample injection is pH 2.3 0.2.
For native samples, be sure that all proteins have been removed before analysis.
PHYSIOLOGIC FLUID SAMPLES:
PREPARATION USING SERAPREP OR URIPREP
Use SERAPREP for preparing serum and other samples with a high buffering capacity, e.g. sardine oil. Use
URIPREP for preparing urine and other samples with low buffering capacity, such as fruit juices, musts and
warts. The efficiency of protein precipitation and the need for post-centrifugation pH adjustment of the sample
determine which reagent is best for your particular sample.
1. In a microcentrifuge tube thoroughly mix equal portions of sample and SERAPREP or URIPREP.
2. Let stand for 5 minutes. Centrifuge the mixture at 13,000 rpm for 5 minutes. Check the supernate pH to
ensure that the range is pH 2.30.2. Adjust the initial mixing ratio as necessary.
3. Filter the supernate with a syringe filter (0.2 or 0.45 um).The filtrate is ready to be injected into an autosampler vial for amino acid analysis.
4. If further dilution is needed, use Li 220 to adjust the concentration of analyte.
Reagent Preparation
TRIONE PREPARATION
TRIONE reagent requires little to no preparation, depending on what type you use.
T100: The one-part TRIONE (Cat. No. T100C) requires no preparation - simply pour the TRIONE directly
into the reagent reservoir and put the cap on the reservoir.
T200: To prepare two-part TRIONE (Cat. No. T200), pour Bottle 1 into the reservoir, add Bottle 2 to the
reservoir, and cap tightly under Nitrogen. Swirl until homogeneous.

6.2-4
Section 6
A pplications
Note: TRIONE is air sensitive, and must be kept under Nitrogen. The useful lifetime of T100 is three months*
unopened, and one month in the reservoir. The shelf-life of T200 is one year* unmixed, and one month in the
reservoir.
*From date of manufacture.
OPA PREPARATION
1. Pour 945ml of the OPA Diluent (Cat.No. OD104) into the reagent reservoir. Save approximately 5 ml for
Step 5.
2. Put the cap on the bottle, open the vent valve, and turn on the gas supply. Thoroughly deaerate the contents
by sparging with inert gas. Continue bubbling for at least 10 minutes.
3. Dissolve 300mg of OPA (Cat. No. O120) in 10mL of HPLC-grade methanol in a clean, dry container.
4. Turn off the gas supply and remove the cap from the bottle. Add the OPA solution to the deoxygenated diluent
in the reservoir. Wash any residual mixture into the reservoir with an additional 12 ml of methanol.
5. Dissolve 2g of Thiofluor (Cat. No. 3700-2000) in the reserved 5 ml of OPA Diluent and add to the
reservoir.
6. Add 3ml of 30% Brij-35 (Sigma) solution.
7. Replace the cap and close the vent valve. Gently swirl the reagent to complete the mixing. Turn on the inert gas.
Note: OPA reagent is sensitive to air oxidation and will degrade over time. The Pinnacle PCX system is designed
to minimize this oxidation. When the OPA reagent reservoir is maintained under inert gas pressure, the OPA
reagent can maintain its activity for up to one week without significant loss of activity.
Post Column Conditions

These are the recommended post-column conditions for the most common methods of amino acid analysis.
For the HPLC conditions, refer to the section titled Sample Chromatograms and Gradient Programs.
USING TRIONE NINHYDRIN REAGENT:
Reagent 1:
TRIONE (Cat. No. T100 or T200)
Pump 1 Flow Rate: 0.30 ml/min
Reactor 1 Volume: 500 l
Reactor 1 Temp:
130C
USING OPA REAGENT:
Reagent 1:
o-Phthalaldehyde and Thiofluor in OD104 Diluent, plus 35% Brij-35
Reactor 1 Temp:
45C

6.2-5
Section 6
A pplications
USING SODIUM HYPOCHLORITE, FOLLOWED BY OPA REAGENT:

Reagent 1:
250 l of 5% Sodium Hypochlorite in GA116 Diluent
Reagent 2:
o-Phthalaldehyde and Thiofluor in OD104 Diluent, plus 30% Brij-35
Reactor 1 Temp:
55C
Reactor 2 Temp:
Ambient
Procedure
Pickering Laboratories recommends different gradient conditions depending on the column and type of sample.
Use the program recommended on the column data sheet for the initial testing. Do not change this
program until you are sure that the other aspects of the system are functioning properly.
The column oven temperature programming gives additional flexibility when optimizing methods. Using
temperature gradient allows to improve separation, shorten analysis time and fine-tune the method for detecting
compounds of interest. Please refer to page 4.8 for details on how to set up timetable for the column oven.
Set the maximum pressure limit on the HPLC to 220 bar to protect the column. Allow the column to equilibrate
for about 30 minutes under initial conditions. Inject 10l of the Amino Acid Standard, and collect three
chromatograms. The first chromatogram will not be representative of the systems performance, so use the
second two to evaluate the performance.

6.2-6
Section 6
A pplications

METHOD 1: 0354675T 70-MIN PHYSIOLOGICAL FLUID LITHIUM (4.6 x 75mm)
GUARD COLUMN: GARD
COLUMN TEMP: SEE GRADIENT
HPLC FLOW RATE: 0.55ml/min
REAGENT FLOW RATE: 0.5ml/min
INJECTION VOLUME: 10ul
HPLC PROGRAM
1700-1125 %
Li365 %
Li375 %
100
0
0
100
0
0
40
60
0
0
100
0
0
100
0
0
0
100
0
0
100
0
0
70
0
0
70
100
0
0
Time
0
10
19
32
43
43.1
57
57.1
72
72.1
COLUMN OVEN PROGRAM

Time
Temp C
0
34
6
34
17
65
25
70
70
70
71
34
RG003 %
0
0
0
0
0
0
0
30
30
0
20
23
15
45
17
16
18 21
2
11
22
44
10
9
36
24
25
19
37
30
26 27
35
13
28 29
12
10
14
20
30
40
40
34
32
31
39
38
43
33
41 42
50
60
70
min
PEAK IDENTIFICATION
1 Phosphoserine
2 Taurine
3 Phosphoethanolamine
4 Urea
5 Aspartic acid
6 Hydroxyproline
7 Threonine
8 Serine
9 Asparagine
10 Glutamic acid
11 Glutamine
12 Sarcosine
*Internal Standard
13 -Aminoadipic acid
14 Proline
15 Glycine
16 Alanine
17 Citrulline
18 -Amino-n-butyric acid
19 Valine
20 Cystine
21 Methionine
22 Allo-Isoleucine
23 Cystathionine
24 Isoleucine
25 Leucine
26 Tyrosine
27 Phenylalanine
28 -Alanine
29 -Amino-i-butyric acid
30 Homocystine
31 -Aminobutyric acid
32 Tryptophan
33 Ethanolamine
34 Ammonia
35 Hydroxylysines
36 Ornitine
37 Lysine
38 1-Methylhistidine
39 Histidine
41 Anserine
42 Carnosine
43 Arginine
44 Glucosaminic Acid*
45 2-Aminoethyl-cysteine (AEC)*
NOTE: This method utilizes column temperature gradient. Use Pinnacle PCX column oven or HPLC column oven with temperature
gradient capabilities.

6.2-7
Section 6
A pplications
METHOD 2: 0354100T ISOTHERMIC PHYSIOLOGICAL FLUID LITHIUM (4.0 x 100mm)

GUARD COLUMN: GARD
COLUMN TEMP: 37C
Time (Min)
0
8
46
86
90
115
122
122.1
140
CONDITIONS
Li750 %
RG003 %
0
0
0
0
35
0
100
0
100
0
94
6
94
6
0
0
0
0
Li275 %
100
100
65
0
0
0
0
100
100
Comment
Inject
Isocratic
Linear Gradient
Linear Gradient
Isocratic
Linear Gradient
Isocratic
Step Change
Re-equilibration
2
1
44
22
40
20
39
17 18
16
19
10
15
41
30
21
31
23
24
26 27
12 13
37
28
35
29
38
43
33
11
45
42
25
34
32
36
14
20
40
60
80
100
120
PEAK IDENTIFICATION
1. Phosphoserine
2. Taurine
3. Phosphoethanolamine
4. Urea
5. Aspartic acid
6. Hydroxyproline
7. Threonine
8. Serine
9. Aspargine
10. Glutamic acid
11. Glutamine
12. Sarcosine
13. -Aminoadipic acid

14. Proline
15. Glycine
16. Alanine
17. Citrulline
18. -Amino-n-butyric acid
19. Valine
20. Cystine
21. Methionine
22. Cystathionine
23. Isoleucine
24. Leucine
25. Norleucine
26. Tyrosine
27. Phenylalanine
28. -Alanine
29. -Amino-i butyric acid
30. Homocystine
31. -Aminobutyric acid
32. Tryptophan
33. Ethanolamine
34. Hydroxylysines
35. Ammonia
36. Creatinine
37. Ornithine
38. Lysine
39. Histidine
40. 3-Methylhistidine
42. Carnosine
43. Anserine
44. -Amino-
guanidinopropionic acid
45. Arginie

6.2-8
Section 6
A pplications
METHOD 3: 0354100T TEMP GRADIENT PHYIOLOGICAL FLUID LITHIUM (4.0 x 100mm)

GUARD COLUMN: GARD
Time (Min)
0
15
27
45
60
60.1
78
78.1
95
95.1
115
CONDITIONS
Li365 % Li375 %
0
0
0
0
60
0
100
0
100
0
0
100
0
100
0
70
0
70
0
0
0
0
1700-1125 %
100
100
40
0
0
0
0
0
0
100
100
RG003 %
0
0
0
0
0
0
0
30
30
0
0
Comment
Inject
Isocratic
Linear Gradient
Linear Gradient
Isocratic
Step Gradient
Isocratic
Step Gradient
Isocratic
Step Gradient
Equilibration
COLUMN OVEN PROGRAM

Time (Min) Temp. C
0
34
13
34
30
65
67
66
80
70
97
70
98
34
1
20
22
16
15 17
18
35
19 21
36
7
23 24
3 43
10
29
34
39
32
9
13
31
33
42
30
11
4
37 38
25 26
12
20
40
41
27 28
14
40
60
80
min
PEAK IDENTIFICATION
1 Phosphoserine
2 Taurine
3 Phosphoethanolamine
4 Urea
5 Aspartic acid
6 Hydroxyproline
7 Threonine
8 Serine
9 Asparagine
10 Glutamic acid
11 Glutamine
12 Sarcosine
13 a-Aminoadipic acid
14 Proline
15 Glycine
16 Alanine
17 Citrulline
18 a-Amino-n-butyric acid
19 Valine
20 Cystine
21 Methionine
22 Cystathionine
23 Isoleucine
24 Leucine
25 Tyrosine
26 Phenylalanine
27 b-Alanine
28 b-Amino-i-butyric acid
29 Homocystine
30 g-Aminobutyric acid
31 Tryptophan
32 Ethanolamine
33 Ammonia
34 Hydroxylysines
35 Ornitine
36 Lysine
38 Histidine
40 Anserine
41 Carnosine
42 Arginine
43 Glucosaminic Acid*
*Internal Standard
NOTE: This method utilizes column temperature gradient. Use Pinnacle PCX column oven or HPLC column oven with
temperature gradient capabilities.
6.2-9
Section 6
A pplications
METHOD 4: 0393250 ISOTHERMIC PHYSIOLOGICAL FLUID LITHIUM (3.0 x 250mm)

GUARD COLUMN: GARD
COLUMN TEMP: 40C
Time(min)
0
17
65
128
145
185
185.1
210
%Li275
100
100
65
0
0
0
100
100
%Li750
0
0
35
100
100
94
0
0
%RG003
0
0
0
0
0
6
0
0
Comment
inject
isocratic
linear gradient
linear gradient
isocratic
linear gradient
step change
re-equilibration
1
2
3
44
22
39
20
5
7
16
18
17
19
23
24
21
12
41
30
15
10
31
25
26 27
13
29
28
35
45
42
43
32
36
14
20
37 38
33 34
11
4
40
40
60
80
100
120
140
160
180 min
PEAK IDENTIFICATION
1. Phosphoserine
2. Taurine
3. Phosphoethanolamine
4. Urea
5. Aspartic acid
6. Hydroxyproline
7. Threonine
8. Serine
9. Aspargine
10. Glutamic acid
11. Glutamine
12. Sarcosine
13. -Aminoadipic acid

14. Proline
15. Glycine
16. Alanine
17. Citrulline
18. -Amino-n-butyric acid
19. Valine
20. Cystine
21. Methionine
22. Cystathionine
23. Isoleucine
24. Leucine
25. Norleucine
26. Tyrosine
27. Phenylalanine
28. -Alanine
29. -Amino-i butyric acid
30. Homocystine
31. -Aminobutyric acid
32. Tryptophan
33. Ethanolamine
34. Hydroxylysines
35. Ammonia
36. Creatinine
37. Ornithine
38. Lysine
39. Histidine
42. Carnosine
43. Anserine
44. -Amino-guanidinopropionic acid
45. Arginie

6.2-10
Section 6
A pplications
METHOD 5: 0354100T PKU AND MSUD SCREENING LITHIUM (4.0 x 100mm)

GUARD COLUMN: GARD
COLUMN TEMP: 38C

10
15
20
Phenylalanine
Comment
Inject
Linear gradient
Step gradient
Isocratic
Step gradient
Re-equilibration
Tyrosine
Norleucine
Isoleucine
%RG003
0
0
100
100
0
0
Leucine
Methionine
%Li750
14
27
0
0
14
14
Cystathionine
%Li275
86
73
0
0
86
86
a-Amino-n-Butyric Acid
Valine
Time (min)
0
25
25.1
30
30.1
42
25
min
6.2-11
Section 6
A pplications
METHOD 6: 1154110T OXIDIZED HYDROLYSATE SODIUM (4.6X110 MM)

GUARD COLUMN: GARD
Time
0
4.0
15.0
16.0
31.0
31.1
33.0
33.1
40
% Na270
100
100
0
0
0
0
0
100
100
HPLC PROGRAM
% Na425 % Na640
0
0
0
0
100
0
0
100
0
100
0
0
0
0
0
0
0
0
COLUMN OVEN PROGRAM

Time
Temp C
0
55
12
55
17
70
32
70
33
55
% RG011
0
0
0
0
0
100
100
0
0
17
6
21
8
14
15
10
22
13
12
11
16
1 2 3
20
18
5
10
15
20
25
30
min
PEAK IDENTIFICATION
1 Aspartic Acid
2 Threonine
3 Serine
4 Glutamic Acid
5 Proline
6 Glycine
7 Alanine
8 Cystine
9 Valine
10 Methionine
11 Isoleucine
12 Leucine
13 Norleucine
14 Tyrosine
15 Phenylalanine
16 Lysine
17 Histidine
18 Ammonia
19 Tryptophan
20 Arginine
21 Cysteic Acid
22 Methionine Sulfone
23 Methionine Sulfoxide
24 Trans-4-Hydroxy-L-Proline
25 D,L & allo-Hydroxylysine
NOTE: This method utilizes column temperature gradient.

Use Pinnacle PCX column oven or HPLC column oven with
temperature gradient capabilities.

6.2-12
Section 6
A pplications
METHOD 7: 1154110T PROTEIN HYDROLYSATE SODIUM (4.6X110 MM)

GUARD COLUMN: GARD
HPLC PROGRAM
% Na315
% Na425
% Na640
100
0
0
100
0
0
0
100
0
0
0
100
0
0
100
0
0
0
0
0
0
100
0
0
100
0
0
Time
0
4.0
15.0
16.0
31.0
31.1
33.0
33.1
40
COLUMN OVEN PROGRAM

Time
Temp C
0
46
4
46
9
70
32
70
33
46
% RG011
0
0
0
0
0
100
100
0
0
17
2 3
16
15
10
9
12
11 1314
20
19
18
5
0

10
15
20
25
30
6.2-13
Section 6
A pplications
METHOD 8: 1154110T COLLAGEN HYDROLYSATE SODIUM (4.6X110 MM)

GUARD COLUMN: GARD
HPLC PROGRAM
% Na315
% Na425
% Na640
100
0
0
100
0
0
0
100
0
0
0
100
0
0
100
0
0
0
0
0
0
100
0
0
100
0
0
Time
0
4.0
15.0
16.0
31.0
31.1
33.0
33.1
40
COLUMN OVEN PROGRAM

Time
Temp C
0
42
4
42
9
70
32
70
33
42
% RG011
0
0
0
0
0
100
100
0
0
17
23
24
1 23
15
8
10
9
16
25
12
14
11
13
20
18
10
15
20
25
30

6.2-14
Section 6
A pplications
METHOD 9: 1154150T ISOTHERMIC OXIDIZED HYDROLYSATE SODIUM (4.0 x 150mm)

GUARD COLUMN: GARD
COLUMN TEMP: 50C
CONDITIONS FOR OXIDIZED SAMPLES

Time (Min)
Na270 %
Na740 %
0
100
0
14
100
0
32
20
80
32.1
0
100
56
0
100
56.1
0
0
58
0
0
58.1
100
0
70
100
0
1154150T
RG011 %
0
0
0
0
0
100
100
0
0
8
9
11
23
20
18
13
14
24 4 5
3
16
19
22
7
0
10
20
30
40
PEAK IDENTIFICATION
3
Aspartic Acid
4 Threonine
5 Serine
6
Glutamic Acid
7 Proline
8 Glycine
9 Alanine
11 Valine
13 Isoleucine

14 Leucine
16 Phenylalanine
18 Lysine
19 Ammonia
20 Histidine
22 Arginine
23 Cysteic Acid
24 Methionine Sulfone
50
min
6.2-15
Section 6
A pplications
METHOD 10: 1154150T ISOTHERMIC PROTEIN AND COLLAGEN HYDROLYSATE SODIUM (4.0 x 150mm)
GUARD COLUMN: GARD
COLUMN TEMP: 48C
CONDITIONS FOR PROTEIN &

COLLAGEN HYDROLYSATE SAMPLES
Time (Min)
Na315 %
Na740 %
RG011 %
0
100
0
0
10
100
0
0
30
0
100
0
53
0
100
0
53.1
0
0
100
55
0
0
100
55.1
100
0
0
67
100
0
0
10
9
11
9 12
34
18
1314
10
1516
17
15
16
20
18
13
14
5
34
*Protein
Hydrolysate
20
1112
*Collagen
Hydrolysate
19
19
22
22
7
2
21
7
0
10
20
30
40
50
min
10
20
30
40
PEAK IDENTIFICATION
1 Methionine-D,L,-Sulfoxide
2 trans-4-Hydroxy-L-Proline
3
Aspartic Acid
4 Threonine
5 Serine
6
Glutamic Acid
7 Proline
8 Glycine
9
Alanine
10 Cystine
11 Valine
12 Methionine
13 Isoleucine
14 Leucine
15 Tyrosine
16 Phenylalanine
17 D,L & allo-Hydroxylysine
18 Lysine
19 Ammonia
20 Histidine
21 Tryptophan
22 Arginine

50
min
6.2-16
Section 6
A pplications
METHOD 11: 1154150 ISOTHERMIC PROTEIN AND COLLAGEN HYDROLYSATE SODIUM (4.0 x 150mm)
GUARD COLUMN: GARD
COLUMN TEMP: 48C
Time(min)
0
12
34
53
53.1
55
55.1
67
%1700-0112*
100
100
0
0
0
0
100
100
%Na740
0
0
100
100
0
0
0
0
%RG011
0
0
0
0
100
100
0
0
Comment
inject
isocratic
linear gradient
isocratic
step gradient
isocratic
isocratic
re-equilibration
Collagen Hydrolysate
11
9
12 13
18
14
5
3 4
20
8
10
6
15
16
19
17
21
7
2
10
20
30
40
50
Protein Hydrolysate
10
9
18
11
8
5
12 13
3 4
14
20
22
15 16
19
21
7
MINUTES
10
20
* For use with columns with serial numbers after 1314

30
40
50
60
6.2-17
Section 6
A pplications
METHOD 12: 1193250 ISOTHERMIC PROTEIN HYDROLYSATE SODIUM (3.0 x 250mm)

GUARD COLUMN: GARD
COLUMN TEMP: 48C
Time(min)
0
10
32
56
56.1
58
58.1
70
%Na328
100
100
0
0
0
0
100
100
%Na740
0
0
100
100
0
0
0
0
%RG011
0
0
0
0
100
100
0
0
11
18
Comment
inject
isocratic
linear gradient
isocratic
step gradient
isocratic
step change
re-equilibration
13
14
20
10
19
8
6
9
12
15
16
22
21
10
20
30
40
50
60
MINUTES
PEAK IDENTIFICATION
1. Methionine sulfoxide
2. Hydroxyproline
3. Aspartic acid
4. Threonine
5. Serine
6. Glutamic acid
7. Proline
8. Glycine
9. Alanine
10. Cystine
11. Valine
12. Methionine
13. Isoleucine
14. Leucine
15. Tyrosine
16. Phenylalanine
17. Hydroxylysines
18. Lysine
19. Ammonia
20. Histidine
21. Tryptophan
22. Arginine
23. Cysteic acid
24. Methionine Sulfone
25. Norleucine

6.2-18
Section 6
A pplications
Precautions for Amino Acid Analysis

Use Pickering Laboratories reagents and eluants. The quality of the chemicals is excellent, and the cost is low
relative to the worth of your analytical results.
Use the Pickering column and eluants. They are designed to work together.
Use the proper start-up and shutdown procedures consistently (see Chapter 2 and 4).
Avoid touching the interior of the mobile phase reservoirs and the dip tubes with your skin. Amino acids in
fingerprints will cause contamination. Gloves are suggested.
When switching a system between ion-exchange and reversed-phase applications, be sure to flush the HPLC
and injector with water before connecting the column. Eluants for one analysis may damage the column for the
other.
Always protect the analytical column by use of the guard column. Always filter the samples through 0.45 m
filter before injecting.
Daily check for leaks at the column fittings; the eluants can be corrosive.
Do not operate with a column pressure above 2800 psi (193 bars) for an extended period of time. Isolate the
source of the high pressureguard column, analytical column, or in-line filter (if in use) and replace
items causing the increased back pressure.
Note: Back-pressure from filter and guard column should be < 36 bars.
During shutdown, flush the column with regenerant for 1520 min. Store the column in regenerant.
When removing the column, rinse the end-fittings with water then plug the column to prevent corrosion.
Contamination usually occurs on the guard column first. Wash it separately from the analytical column. This
will save much time in the washing and re-equilibration.
Contaminants to be especially wary of: iron and other polyvalent cations, organic dyes, lipids, surfactants, and
detergents. These may cause irreversible damage.
Organic solvents will cause the resin in the column to swell leading to high back-pressure and broadened
peaks. The column sometimes can be regenerated.
6.2-19
Section 6
A pplications
Always wear gloves during the preparation of the reagents. The OPA and Thiofluor can cause skin irritation.
Also fingerprints can cause contamination of the reagent. TRIONE will stain skin.
The OPA reagent is sensitive to air oxidation, degrades over time, and should be prepared fresh for optimum
sensitivity. OPA reagent is stable for at least one week when pressurized with inert gas.
Thiofluor is extremely hygroscopic. Always keep in a tightly closed container.
The preparation of the OPA Diluent by the user is not recommended because sodium borate (any grades)
contains excessive amounts of heavy metal contaminants and insoluble matter. These impurities will eventually
precipitate in the reactor and flowcell. The one year warranty does not cover damage caused by these
contaminants.
The pre-mixed TRIONE has a shelf life of 3 months*. As it ages, the risk of precipitate formation increases.
Using outdated TRIONE is a major cause of clogging in post-column systems.
Never put new Trione in the bottle containing old reagent. This will cause premature aging of reagent. Always
discard old reagent and clean the bottle before putting new TRIONE in.
As TRIONE ages, the color intensity for primary amines increases by up to 20%. A small drop in sensitivity
when changing to a new lot of TRIONE is not unusual.
Air oxidation of TRIONE causes the intensity for primary amines to decrease, but does not affect the intensity
for secondary amines. This makes secondary amines appear bigger. Also the reagent becomes more yellow
when it is oxidized.
Frequently observe and record the pressures and check for leaks. You may find a problem before it becomes
serious.
Do not operate the heated reactor above the boiling point of the eluant unless the back-pressure regulator
is connected to the waste line of the detector. Boiling inside the reactor can cause precipitates to block the
reactor. Operating above the boiling point without a back pressure regulator will void your warranty.
Note: Before making any change in the gradient, temperature, or other operating conditions, get at least two
chromatograms in a row with the same problem. After you make a change, get at least two chromatograms
showing the same effect of the change. This is especially true when you are trying to optimize gradient
conditions.

6.2-20
Section 6
A pplications
Make only one small change at a time.

Make a change only after you have collected at least two chromatograms showing the same separation. This
usually means three injections, as the first injection of a series rarely is representative of the rest of the series.
Optimize the separations in the early part of the gradient before optimizing the late part.
Every model of HPLC forms gradients differently. The programs suggested in this manual or in the information
sheets are typical of the more popular HPLC pumps. Consult Pickering Laboratories if you need advice.
If you need only the early part of the chromatogram, you can save time by truncating the gradient. Go to the
final concentration of regenerant and hold it until the most basic component (arginine) elutes, then
re-equilibrate with the initial buffer.
The separation is temperature sensitive. Adjusting the temperature may improve it. For example, the resolution
of threonine and serine improves when the column temperature is cooler, however the resolution
of tyrosine and phenylalanine is best when the column temperature is warmer.
Surfactants, dyes, ninhydrin, and lipids usually cannot be removed. Prevention is the only cure.

6.2-21
Section 6
A pplications
Notes

6.2-22
Section 6
A pplications
Notes

6.3
Section 6
A pplications
CARBAMATES
6.3 Introduction
6.3 Background
6.3-5 Post-column Conditions
6.3-5 Analytical Procedure
6.3-5 Sample Chromatograms and Gradient Programs
6.3-12 Precautions
6.3-13 Notes
Introduction
High-performance liquid chromatography (HPLC) with post-column derivatization is a technique for rendering
analytes more detectable than they would otherwise be in their native forms. Post-column derivatization can
give improved sensitivity or better selectivity (reduction of interference) leading to lower detection limits.
The Pickering Laboratories Pinnacle PCX was developed to facilitate the determination of carbamate
insecticides, meeting or exceeding performance requirements of U.S. Environmental Protection Agency
(USEPA) Method 531.1/531.2, and the AOAC International Protocol 29.A05:
High sensitivity: detection limits of 0.10.5ng (or 0.21ppb levels for drinking water) can be routinely
achieved.
Selectivity (specificity): only N-methylcarbamates and N-methyl carbamoyloximes plus components reactive
to OPA under the specified operating conditions are detected.
Minimum sample preparation: drinking water can be directly injected into the HPLC after filtration. No pre extraction or sample cleanup is required.
The analysis is easily automated for unattended analyses with the addition of an autosampler.
There are a number of carbamate pesticide compounds employed worldwide which are not included in the 11
compounds mandated by USEPA Method 531.1/531.2 and AOAC Protocol 29.A05. The Pickering Laboratories
Carbamate column can separate as many as 23 compounds.
Background
Carbamates, a class of highly effective commercial insecticides, are used worldwide to protect crops from insect
pests. Applied directly to food crops such as grains, fruit, and vegetables, carbamates may seep into drinking
water sources through agricultural runoff. In addition, if food crops are harvested too soon after application,

6.3-1
Section 6
A pplications
residues of carbamates and their by-products may remain in the produce. The use of carbamate insecticides
has created a requirement for a simple, reliable, and sensitive method of residue analysis for these compounds
found in vegetable matter, drinking water, and industrial waste-water. The USEPA Methods 531.2 and 531.1,
and the AOAC International protocol 29.A05, describe a direct-inject method which employs gradient liquid
chromatography with fluorescence detection, accomplished by post-column hydrolysis and derivatization of the
eluted carbamates.
The general structure of the carbamate insecticides is an N-methyl substituted urethane with the variation in the
ester moiety. The structural formulas are shown in Figure 6.3-A.
H3C
CH3
H3C
CH3
S
O
H3C
NH
N
S
CH3
CH3
NH
Aldicarb Sulfoxide
CH3
H3C
HN
Aldicarb Sulfone
Carbaryl
H3C
CH3
CH3
H3C
NH
NH
HN
O
CH3
H3C
Propoxur
CH3
Oxamyl
CH3
Carbofuran
CH3
HN
CH3
H3C
Methiocarb
CH3
CH3
1-Naphthol
NH
HN
OH
CH3
S
CH3
CH3
CH3
CH3
OH
NH
H3C
3-Hydroxy Carbofuran
Methomyl
Aldicarb
CH3
CH3
H3C
CH3
HN
CH3
S
H3C
CH3
CH3
Br
BDMC
Figure 6.3-A

6.3-2
Section 6
A pplications
The separation of the 12 carbamates shown in Figure 6.3-A is achieved with the Pickering Carbamate Column
maintained at constant temperature and a water/methanol gradient. The carbamates elute principally in
relation to their relative hydrophobicity. Aldicarb sulfone, which is minimally hydrophobic, elutes early while
methiocarb, which is more hydrophobic, elutes towards the end of the gradient.
The separated carbamates are first hydrolysed by sodium hydroxide (NaOH) at 100C to release an alcohol,
carbonate, and methylamine. In the second post-column reaction, methylamine reacts with o-phthalaldehyde
(OPA) and the nucleophilic Thiofluor to form a highly fluorescent isoindole derivative (Figure 6.3-B).
O
1.
NH
CH3
OH-
H2O
CH3NH2
OH
CO32
100C
Carbamate
CH3
N
HS
N
CHO
2.
+
CHO
OPA
CH3
CH3NH2
CH3
CH3
CH3
pH>9
Fluorescent Isoindole
Figure 6.3-B
Note: 1-naphthol fluoresces without derivatization and the hydrolysis of carbaryl in the post-column reactor
also produces 1-naphthol, but at a different retention time. This observation is useful for troubleshooting.

The following is a general sample preparation for Water samples containing carbamate. For details, consult the
EPA 531.2 method for drinking water and the AOAC method for Vegetables.

6.3-3
Section 6
A pplications
FOR WATER SAMPLES

Sampling Protocol
To preserve the Carbamates in water, this procedure should be carried out in the field.
SAMPLING PROTOCOL for EPA Method 531.1 and 531.2
1. Add 1.8 ml of ChlorAC Buffer to each pre-cleaned 60 ml sample vial (see note about well and river waters!)
2. If the water sample is chlorinated, dechlorinate with 5 mg of Sodium thiosulfate per 60 ml sample.
3. Fill the sample vials with the dechlorinated water, seal, and mix well.
4. Refer to method EPA 531 or 531.2 for sample transportation and storage details.
Sample Preparation
Filter 2ml of sample through a 0.45m filter.
Inject 200-400l.
FOR STANDARDS AND BLANKS
Use 10 ml ChlorAC Buffer diluted to 1000 ml with HPLC-grade water.
Note: Well and river waters contain colloidal iron which would dissolve if samples are preserved prior
to filtration only to precipitate out again as the hydroxide in the reactor. For well and river waters, it is
recommended to filter the water first through a 0.45m filter, and then preserve with ChlorAC.
Reagent Preparation
The two derivatization reagents required for carbamate analysis are a hydrolysis reagent (NaOH) and
o-phthalaldehyde reagent.
Note: During initial installation, the reagent bottles, lines, and pump should first be cleaned and primed with
methanol to reduce possible fluorescence background.

6.3-4
Section 6
A pplications
REAGENT 1, HYDROLYSIS REAGENT

Turn off the inert gas.
Thoroughly wash the two reagent reservoirs and then rinse with methanol. Wipe down the dip tubes with
methanol and a clean cellulose tissue.
The hydrolysis reagent does not require preparation. Pour the hydrolysis reagent (Cat. No. CB130 or CB130.2)
directly into the reagent reservoir for Reagent 1. It should be labeled Hydrolysis Reagent. Put the cap on the
reservoir. Close the vent valve.
The Hydrolysis reagent remains stable indefinitely.
Note: The preparation of the Hydrolysis Reagent by the user is not recommended because it is hard to obtain
NaOH of adequate purity.
REAGENT 2, OPA REAGENT
1. Pour 945ml of the OPA Diluent (Cat. No. CB910) into the reagent reservoir. Save approximately 5ml
for step 5.
2. Put the cap on the bottle, open the vent valve, and turn on the gas supply. Thoroughly de-aerate the contents
by sparging with inert gas. Continue bubbling for at least 10 minutes
3. Dissolve 100 mg of OPA (Cat. No. O120) in approximately 10 ml of HPLC-grade methanol in a clean, dry
container.
4. Turn off the gas supply and remove the cap from the bottle. Add the OPA solution to the deoxygenated
Diluent in the reservoir.
5. Dissolve 2 g of Thiofluor (Cat. No. 3700-2000) in the reserved 5 ml of the OPA diluent from Step 1 and add
into the reservoir.
6. Replace the cap and turn on the gas flow. Continue sparging for another minute. Close the vent valve. Gently
swirl the reagent to complete the mixing.
Note: The preparation of the OPA Diluent by the user is not recommended because sodium borate (any
grades) contains excessive amounts of heavy metal contaminants and insoluble matter. These impurities will
eventually precipitate in the reactor and flowcell. The one year warranty does not cover damage caused by these
contaminants.


6.3-5
Section 6
A pplications
The OPA reagent is sensitive to air oxidation and degrades over time. When the OPA reagent reservoir is
maintained under inert gas pressure, the OPA reagent maintains its activity for one week without significant
loss of activity.
Post-column Conditions
These are the recommended post-column conditions for carbamate analysis. For the HPLC conditions, refer to
the section titled Sample Chromatograms and Gradient programs.
Reagent 1: CB130 or CB130.2, Hydrolysis Reagent (NaOH)
Reagent 2: o-Phthalaldehyde and Thiofluor in CB910 Diluent
Reactor 1 Temp: 100C
Reactor 2 Temp: Ambient
Analytical Procedure
Allow the column to equilibrate for about 20 minutes under initial conditions.
Inject 10l of Carbamate Text Mixture (or the appropriate volume of your standard), and collect the first
chromatogram.

The Peak Names apply to all chromatograms in this section.

1.
2.
3.
4.
5.
6.
Aldicarb sulfoxide (Standak)

Aldicarb sulfone
Oxamyl (Vydate)
Methomyl (Lannate)
3-Hydroxy carbofuran
Aldicarb (Temik)

7. Propoxur (Baygon)
8. Carbofuran (Furadan)
9. Carbaryl (Sevin)
10. 1-Naphthol
11. Methiocarb (Mesurol)
12. BDMC internal standard
6.3-6
Section 6
A pplications
METHOD 1: 0840250 C8 COLUMN (4.0X250MM) WATER/METHANOL GRADIENT WITH METHANOLIC AND

AQUEOUS SAMPLES
HPLC FLOW RATE: 0.8ML/MIN
COLUMN TEMPERATURE: 50 C
REAGENT FLOW RATE: 0.3ML/MIN
CONDITIONS FOR WATER/METHANOL GRADIENT

Time (Min)
Water %
MeOH %
0
85
15
2
85
15
42
30
70
46
30
70
46.1
0
100
51
0
100
51.1
85
15
59
85
15
Carbamate Test Mix, 2.5ppm, 10ul injection, 25cm, Carbamate C8 column (0840250)
6
2
3
7
5
10
11
10
min
40
30
20
Carbamate Test Mix, 0.25ppb, 100ul injection, 25cm, Carbamate C8 column (0840250)
1
4
2
3
9
5
7
8
N
10
20
30
10
11
min
40

6.3-7
Section 6
A pplications
METHOD 2: 0840250 C8 COLUMN (4.6X110 MM) WATER/ACETONITRILE GRADIENT WITH METHANOLIC

AND AQUEOUS SAMPLES
COLUMN TEMP: 50 C
CONDITIONS FOR WATER/ACETONITRILE GRADIENT

Time (Min)
Water %
ACN %
0
90
10
90
10
2
46
49
51
46.1
30
70
49
30
70
49.1
90
10
59
90
10
40 pg of Carbamates in 400 L
9
3
11
12
8
10
1.
2.
3.
4.
10
Aldicarb sulfoxide
Aldicarb sulfone
Oxamyl
Methomyl

15
20
25
5.
6.
7.
8.
30
35
3-Hydroxycarbofuran
Aldicarb
Propoxur
Carbofuran
40
45
50
min
9. Carbaryl
10. 1-Naphthol
11. Methiocarb
12. BDMC (internal standard)
6.3-8
Section 6
A pplications
METHOD 3: 1846150 C18 COLUMN (4.6X150MM) WITH METHANOLIC SAMPLES

REAGENT FLOW RATE: 0.3 ML/MIN
CONDITIONS FOR METHANOLIC SAMPLES

Time (Min)
Water %
MeOH %
0
85
15
0.5
85
15
28.5
30
70
28.6
0
100
33.5
0
100
33.6
85
15
41
85
15
Carbamate Test Mix, 2.5ppm, 10l injection, 15 cm, Carbamate C18 column(1846150)
4
9
3
7
5
10
8
10
20
11
min
30

6.3-9
Section 6
A pplications
METHOD 4: 1846150 C18 COLUMN (4.6X150 MM) WITH AQUEOUS SAMPLES

COLUMN TEMP: 42 C
CONDITIONS FOR AQUEOUS SAMPLES

Time (Min)
Water %
MeOH %
0
100
0
1
100
0
1.1
88
12
26
30
70
26.1
0
100
28
0
100
28.1
100
0
38
100
0
4 ng of Carbamates in 150 L
3
5
7
8

10
15
20
11
10
25
12
30
min
6.3-10
Section 6
A pplications
METHOD 5: 0846250 C8 COLUMN (4.6X250MM) WITH METHANOLIC SAMPLES

CONDITIONS FOR METHANOLIC SAMPLES

Time (Min)
Water %
MeOH %
0
85
15
1
85
15
44
25
75
44.1
0
100
49
0
100
49.1
85
15
57
85
15
Carbamate Test Mix, 2.5ppm, 10l injection, 25cm, Carbamate C8 Column (0846250)
4
1
2
3
5
7
10
15
20
25
10
30
11
35
12
40
min

6.3-11
Section 6
A pplications
METHOD 6: 0846250 C8 COLUMN (4.6X250MM) WITH AQUEOUS SAMPLES

CONDITIONS FOR AQUEOUS SAMPLES

Time (Min)
Water %
MeOH %
0
100
0
1
100
0
1.1
82
18
36
30
70
39
30
70
39.1
0
100
41
0
100
41.1
100
0
55
100
0

6.3-12
Section 6
A pplications
Upon completion of the analysis, follow the shutdown procedure described in Section 4. Store the carbamate
column in 100% Methanol
Note: The automatic valves prevent reagents from back-flowing onto the column. The inert gas should be left on
to preserve the OPA reagent.
Precautions for Carbamate Analysis

sensitivity. OPA reagent maintains its activity for up to one week when pressurized with inert gas.
contaminants.
Use HPLC-grade methanol and water (Fisher Scientific, JT Baker, or Merck) for carbamate analysis to avoid
problems with baseline drift, spurious peaks, and noise.
Use bottled HPLC-grade water if possible (Fisher Scientific, JT Baker, or Merck), especially during the initial
system start-up. If water from a water purification system is used, ensure the system has an activated charcoal
unit to eliminate organics, and that the charcoal cartridge is placed after the ion-exchange cartridges. (Many
ion-exchange resins leach out OPA-positive contaminates that cause unacceptable fluorescence background.)
The water in the solvent reservoir should be changed every 3 to 4 days to prevent possible bacterial growth.
The test mixture for carbamate is for qualitative use only. It is not recommended for calibration purposes.
Filter all samples through a 0.45m membrane filter. Some samples may require even more stringent filtration,
especially if colloids are present.
Aqueous samples must always be properly buffered. Consult EPA Methods 531.1 or 531.2 for details.
For carbamate analysis with methanolic samples, inject 10l. Large amount of organic solvents can cause
peak distortion.
For small aqueous sample volumes (< 150l) either of the two Pickering columns can be used. For volumes
greater than 300l, use only the 25cm column. A gradient delay time should be programmed into the analysis
(0% organic) to trap the sample onto the head of the column.
Avoid purging the system with 100% acetonitrile as precipitation of borate salt in the reactor might occur. Do
not exceed 70% acetonitrile if it will be used as the mobile phase.
Do not store the column in water.
Use the Pickering Laboratories carbamate analysis column, which is specifically designed and tested for the
separation of carbamates in the EPA Methods.

6.3-13
Section 6
A pplications
Notes

6.4
Section 6
A pplications
GLYPHOSATE
6.4 Introduction
6.4-1 Background
6.4-4 Analytical and Post-column Conditions
6.4-4 Analytical Procedure
6.4-5 Sample Chromatograms
6.4-6 Precautions
6.4-8 Notes
Introduction
High-performance liquid chromatography (HPLC) with post-column derivatization is a technique for rendering
analytes more detectable than they would otherwise be in their native forms. Post-column derivatization can
give improved sensitivity or better selectivity (reduction of interference) leading to lower detection limits.
The Pickering Laboratories Pinnacle PCX was developed to facilitate the determination of the herbicide
glyphosate (and its metabolite AMPA), meeting or exceeding performance requirements of USEPA Method 547.
The Pickering Post-column method can also be used for the determination of Glyphosate and AMPA in
plants and soils. Pickering has improved sample preparation procedure for vegetable samples. It is a simple
extraction followed by clean-up on a strong cation-exchange cartridge. The procedure is listed later on in this
chapter.

6.4-1
Section 6
A pplications
Background
Glyphosate and AMPA are separated on a strong cation-exchange column (fully sulfonated, cross-linked
polystyrene, mixed K+/H+ form). After isocratic separation, the column is regenerated with dilute KOH, then
re-equilibrated with eluant.
Fluorometric detection follows a two-stage post-column reaction. In the first stage, glyphosate is oxidized
by hypochlorite to glycine. In the second stage, glycine reacts with o-phthalaldehyde and Thiofluor
(a mercaptan) at pH 910 to produce a highly fluorescent isoindole. AMPA does not need the initial oxidation
to react with OPA (Figure 6.4-A); indeed oxidation reduces its fluorescent yield.
1.
O2C
N
H
O2C
OCl-
PO 32-
NH2
CH3
Glycine
Glyphosate
N
CHO
2.
Glycine
CHO
OPA
H 2N
PO 32-
AMPA
CH3
CO 2-
H 2N
pH _> 9
CH3
N
HS
R=
CH2 CO2- for Glycine
R=
CH2 PO32- for AMPA
CH3
Thiofluor
Figure 6.4-A

The following is a suggested basic sample preparation for Vegetable and Water samples containing glyphosate.
The method for Vegetables is different from the procedure called out by the AOAC. We have developed
ion-exchange cartridges, which we have fully qualified in our lab, and which greatly improve the ease and
reproducibility of the extraction while at the same time reducing many of the trouble aspects of the original
published method (e.g. iron contamination).
FOR VEGETABLE SAMPLES
Extraction
To 25g of a homogenous sample add enough water (after estimation of moisture content) to make the total
volume of water 125 ml. Blend at high speed for 3-5 min. and centrifuge for 10 min. Transfer 20 mL of the
aqueous extract into a centrifuge tube and add 15 mL of methylene chloride (to remove nonpolar

6.4-2
Section 6
A pplications
co-extractives). Shake for 2-3 min. and centrifuge for 10 min. Transfer 4.5 mL of the aqueous layer into a vial
and add 0.50 mL acidic modifier solution (16g KH PO , 160 ml H O, 40 ml Methanol, 13.4 ml HCl). Shake and
centrifuge for 10 min.
2
Matrix specific modification

Plants with high: 1) Water 2) Protein 3) Fat Content
1) For crops that absorb large amounts of water, reduce test portion to 12.5g keeping water volume the same.
2) For crops that have high protein content add 100 l HCl to 20 ml aliquot of crude extract. Cap, shake and
centrifuge for 10 min.
3) For crops that have high oil content, do the methylene chloride partition twice.
Cation-exchange cleanup
Transfer 1 mL of extract (representing 0.18g normal crop or 0.09g dry crop) to the column reservoir and elute
to the top of the resin bed. Add 0.70 mL of the elution solution (160 mL H20, 2.7 mL HCl, 40 mL Methanol)
and discard the effluent. Repeat with a second 0.70 mL portion and discard effluent. Elute with
12 mL of the elution solution and collect in a round-bottomed flask. Evaporate to dryness in a water bath set at
40C using a rotary evaporator. Or collect in a centrifuge tube and evaporate using a vacuum vortex evaporator.
Dissolve residue in 2.0 mL of the elution solution (use 1.5 ml for dry crops). Extracts before
evaporation can be stored refrigerated for up to 7 days.
FOR WATER SAMPLES
Filter water through a 0.45m membrane filter, and inject 200-400 ul.
If the glyphosate comes out as a doublet, add 2 drops of Restore directly to the sample vial.
Reagent Preparation
HYPOCHLORITE REAGENT
400
Note: 5% Sodium hypochlorite must be used for preparing

oxidizing reagent (can be obtained from local grocery stores).
300
Peak Area
Pour 945 ml of the Hypochlorite Diluent (GA116) directly into

the reagent reservoir. This should be labeled Oxdizing Reagent.
Add 100 l of 5% sodium hypochlorite solution to the diluent.
The exact amount will depend on the actual hypochlorite
concentration of the stock solution. When you get your first
chromatograms, you will be able to adjust the amount to optimize
the relative peak areas of glyphosate versus AMPA. Figure 6.4-B
shows a typical response curve.
350
Glyphosate
AMPA
250
200
150
100
50
0
0
100
200
300
400
500
L of 5% NaOCl per L of GA116
Figure 6.4-B

6.4-3
Section 6
A pplications
Cap the reservoir, close the vent valve, and swirl the solution to mix it thoroughly.

Note: The hypochlorite concentration slowly decreases with time. This will manifest itself as a change in the
relative peak areas of glyphosate and AMPA. It will remain usable for several days, but we recommend you
calibrate daily.
Caution! Do NOT use calcium hypochlorite in the oxidizing reagent. This will cause plugging of the postcolumn reactor. The one year warranty does not cover damage caused by calcium hypochlorite-based reagents.
The EPA Draft Method 547 is wrong on this point; Ca (PO ) is insoluble in water.
3
OPA REAGENT
1. Pour 945 ml of the OPA Diluent (Cat. No. GA104) into the reagent reservoir. Save approximately 5 ml for
step 5.
2. Put the cap on the bottle, open the vent valve, and turn on the gas supply. Thoroughly de-aerate the contents
by sparging with inert gas. Continue bubbling for at least 10 minutes.
3. Dissolve 100 mg of OPA (Cat. No. O120) in approximately 10 ml of HPLC-grade methanol in a clean, dry
container.
4. Turn off the gas supply and remove the cap from the bottle. Add the OPA solution to the deoxygenated
Diluent in the reservoir.
5. Dissolve 2 g of Thiofluor (Cat. No. 3700-2000) in the reserved 5 ml of the OPA Diluent and add into the
reservoir.
6. Replace the cap and turn on the gas flow. Continue sparging for another minute. Close the vent valve. Gently
swirl the reagent to complete the mixing.
Caution! The preparation of the OPA Diluent by the user is not recommended because sodium borate (any
grades) contains excessive amounts of heavy metal contaminants and insoluble matter. These impurities will
eventually precipitate in the reactor and flowcell. The one-year warranty does not cover damage caused by
these contaminants.
Note: The OPA reagent is sensitive to air oxidation and degrades over time. When the OPA reagent reservoir
is maintained under inert gas pressure, the OPA reagent maintains its activity for up to one week without
significant loss of activity.

6.4-4
Section 6
A pplications
Analytical and Post-column Conditions

These are the recommended conditions for glyphosate analysis using the 1954150 column and 1953020 guard
column.
Column Temperature: 55C
HPLC Flow Rate: 0.4 ml/min
HPLC Program:
Times (min)
0
15.0
15.1
17
17.1
27.0
%K200
100
100
0
0
100
100
%RG019
0
0
100
100
0
0
Comment
Inject
Isocratic
Regeneration
Isocratic
Step Change
Re-equilibration
The exact time of equilibration depends on the internal volume of your HPLC. When the baseline and column
pressure are stable for two minutes, the column has been re-equilibrated.
Post-Column Conditions:
Reagent 1: 100 l of 5% NaOCl in GA116 Diluent
Reagent 2: o-Phthalaldehyde and Thiofluor in GA104 Diluent
Reactor 1 Temp: 36C
Reactor 2 Temp: Ambient
Analytical Procedure
Allow the column to equilibrate for about 20 minutes under initial conditions.
Inject 10l of Glyphosate Text Mixture (or the appropriate volume of your standard), and collect the first
chromatogram.

6.4-5
Section 6
A pplications
Figure 6.4-C shows a typical Glyphosate and AMPA chromatogram. In a standard with Glyphosate and AMPA
at equal concentration, the peak heights should be equal. The peak heights are influenced by the amount of
hypochlorite in Reagent 1.
Sample Chromatograms
Glyphosate Test Mix, 2.5ppm, 10l injection
Glyphosate
AMPA
10
20
min
Figure 6.4-C
Glyphosate and AMPA, 13ppb in K200, 100l injection

Glyphosate
AMPA
10
20
min
Figure 6.4-D

6.4-6
Section 6
A pplications
Broccoli sample spiked with Glyphosate and AMPA, 50 ppb, 100 l injection
Glyphosate
AMPA
10
15
min
Figure 6.4-E
Upon completion of the analysis, follow the shutdown procedure described in Section 4. Store the column in
RG019.
Excessive flushing will require an equally excessive re-equilibration when you start up again.
Note: The automatic valves prevent reagents from back-flowing onto the column. The inert gas should be left on
to preserve the OPA reagent.
Precautions to be Aware of in Glyphosate Analysis

sensitivity. OPA reagent is stable for at least one week when pressurized with inert gas.
contaminants.
Use Restore if Iron contamination of the column is suspected.

6.4-7
Section 6
A pplications
Contamination usually occurs on the guard column. Wash it separately from the analytical column. This will
save much time in the washing and re-equilibration.
Contaminants of special concern: iron and other polyvalent cations, organic dyes, surfactants, detergents, and
lipids. They may cause irreversible damage.
Organic solvents will cause the resin in the column to swell. This leads to high back-pressure and broadened
peaks. The column sometimes can be regenerated.
Use Pickering eluants with the Pickering column, as they are designed to work together.
The test mixture for glyphosate is for qualitative use only. It is not recommended for calibration purposes.
Filter all samples through a 0.45m membrane filter. Some samples may require even more stringent filtration,
especially if colloids are present.
Aqueous samples must always be properly buffered. Consult EPA Method 547 for details.

6.4-8
Section 6
A pplications
Notes

6.4-9
Section 6
A pplications
Notes

7.1
Section 7
T roubleshooting
Section 7
Troubleshooting
7.1 Contact Pickering Laboratories for Support
7.2 Instrument Parameter Log
7.2 Troubleshooting Advice
7.3 Common System Problems
7.4 Common Chromatography Problems
7.6 Common Column Problems
7.7 Application-specific Troubleshooting
Amino Acids
Carbamates
Glyphosate
7.8 Software Troubleshooting
7.11 Procedures
To Remove Silica Deposits From Reactor
To Remove Mineral Deposits In The Reactor From Hard Water
To Remove Grease Deposits
If Reagent Backflows Onto Column
If Organic Solvent is On Cation-Exchange Colunm
If NaOH is On Column
To Remove Iron Contamination From Column
To Pump RESTORE Through The Glyphosate Column
CONTACT PICKERING LABORATORIES FOR SUPPORT

There are several easy ways to contact Pickering Laboratories for Technical Support:
Email: support@pickeringlabs.com
Telephone: 800-654-3330 or 650-694-6700
Fax: 650-968-0749
Web Site: www.pickeringlabs.com
Click on the Support tab to send us an email.
Pickering Laboratories business hours are:
Monday thru Friday, 8 AM to 5 PM, Pacific Standard Time (GMT 08:00)


7.2
Section 7
T roubleshooting
We will ask you a set of standard questions:

What application are you running?
What are the pressures in your Pinnacle PCX system?
What is the brand and model of your HPLC system?
What type of samples are you injecting?
Please email or fax to us a chromatogram
Please email the Log files
INSTRUMENT PARAMETER LOG

If you should have any problems with your Pinnacle PCX, the Instrument Log file is a key resource to finding
the cause of, and solving any problems. Pinnacle PCX records detailed information about the instrument
parameters in the Log files. Pump(s) flow rates and pressures, reagent volume, column and reactor
temperature, s and flags from HPLC are all stored for 4 days before being overwritten. Log files are necessary to
troubleshoot Pinnacle PCX system and software and should always be collected after a problem is detected. To
collect Log files go to Help, select Send Log to Support and select file from the day the problem happened.
Save the file with the date stamp and e-mail it to support@pickeringlabs.com. If you are not sure about the time
collect all 4 log files.
Pickering Laboratories strongly recommends that you record your daily operating pressures, and any
maintenance performed on the instrument. This log will be invaluable to your laboratory for troubleshooting
and problem prevention.
Make copies of the blank form in Appendix and complete the parameter log on the photocopy.
GENERAL TROUBLESHOOTING ADVICE

Rules of Dolan and Snyder [see references]
Rule of One: Make one change at a time.
Rule of Two: Confirm the problem before fixing it.
Substitution Rule: Swap in a good part for a questionable one.
Put it Back: If swapping does not fix it, put the original back in.
Write it Down: Changes or modifications, incidents.
Crystal Ball: Preventive maintenance saves more time in the long run.
Buffer Rule: Remove buffers from LC when not in use.
7.3
Section 7
T roubleshooting
General Procedure for Troubleshooting

Examine the system front to back. Repair all leaks.
Verify that all settings, eluants, reagents, valves, etc. are according to specifications.
Have there been any changes in the system?
Compare against reference conditions: standard sample, column, parameter log as appropriate.
Gather information: observations, manuals, books, technical assistance.
Test your conclusions about the nature of the problem.
Start working.
Before making any change in the gradient, temperature, or other operating conditions, get at least two
chromatograms in a row with the same problem. After you make a change, get at least two chromatograms
showing the same effect of the change. This is especially true when you are trying to optimize gradient
conditions.
COMMON SYSTEM PROBLEMS

OBSERVED PROBLEM
COMMON CAUSE
ACTION TO TAKE
NOTES
Low Reagent pressure
Air in reagent pump

Reagent flow rate too low
Leaking fittings
Check for leaks

Change pump seals
Perform Flush Pump

Tighten leaking fittings
High Reagent pressure
Obstruction of flow path by deposits

Over-tightened fittings
Pinched tubing
Obstruction of detector flow-cell
Defective back-pressure regulator
Determine the exact location of the

blockage.
Disconnect one fitting at a time,
moving backward from the backpressure regulator end, until the
pressure drops
For partial blockage, clean tubing with

solvent/water
For total blockage, replace appropriate
part
Reagent pump stops or delivers wrong

flow rate
Check pump setting

Check reagent pressurization
Check pump seal for leakage
Test or clean valves
continued

7.4
Section 7
T roubleshooting
COMMON SYSTEM PROBLEMS
continued
OBSERVED PROBLEM
COMMON CAUSE
ACTION TO TAKE
NOTES
Over-pressure relief valve is opened
There is a blockage in the system
Determine the exact location of the

blockage.
Disconnect one fitting at a time,
moving backward from the backpressure regulator end, until the
pressure drops
Flush the system with solvent/water

until pressure drops, or replace
appropriate part
Blocked Heated Reactor reactor
Improper Shutdown
Dissolved silica precipitating in the
reactor
Contaminated reagents
Mineral deposits from hard-water
samples or reagents
Greasy samples
Use of calcium hypochlorite
Home-made reagents
Hydrindantin deposits from Expired
TRIONE
Follow the procedure for removing

mineral deposits on Page 7.8
Silica from NaOH backflow onto

column
Use of calcium hypochlorite as the
oxidant in glyphosate determination
Preparing your own reagents with
poor quality chemicals
Follow the procedure for removing

Silica deposits on Page 7.8
Replace heated reactor if flushing does
not help
COMMON CHROMATOGRAPHY PROBLEMS

When having chromatography problems collect 2-3 chromatograms that show the problem. Fax these and most
recent normal one to Pickering Laboratories at (650) 968-0749.
OBSERVED PROBLEM
COMMON CAUSE
ACTION TO TAKE
High Background Signal
Contaminated Eluant
Bacterial Growth
Fingerprints
Contaminated Reagent(s)
Defective chemicals
Flush HPLC system and Pinnacle PCX

with 80/20 Water/MeOH
Put new eluants on HPLC, make new
reagents
Noisy Baseline
Worn pump seal

Detector noise
Chemical contamination
Reagent too old
Check for pattern in the noise.

If the background signal is also
elevated, check for chemical
contamination, or an error in
formulation
NOTES
Match the frequency of the noise to

one of the pumps.
If the noise is random, check the
detector
continued

7.5
Section 7
T roubleshooting
COMMON CHROMATOGRAPHY PROBLEMS
continued
OBSERVED PROBLEM
COMMON CAUSE
ACTION TO TAKE
NOTES
Peaks disappear or diminish
Improper Reagent preparation

Out of Reagent
Reactor at wrong temperature
Reagent flow rate improper
Dirty flowcell
Dirty autosampler
Deteriorated samples
Metal contamination of column due to
poor sample preparation or corrosion
in system
Oxidized TRIONE or OPA
Reagent pump mis-adjusted
Iron contamination of column.
Prepare fresh reagents

Prepare fresh standards from neat
reference material.
Test with a second fluorescent or UV/
VIS detector.
Change the rotor seal of the
autosampler or use a manual
injector.
Flush amino acid column with
regenerant.
Check reactor temperature
Clean the flowcell
Follow the procedure for Iron removal
from the column on Page 7.9
Remove all stainless steel frits from
reservoirs
Clean or replace any corroded parts.
Flush Glyphosate column with Restore
All disappear except 1-naphthol and

carbaryL = OPA reagent expired
All disappear except 1-naphthol = Out
of Hydrolysis Reagent
Varied peak size, some missing =
All peaks diminish = dirty flowcell,
autosampler, or deteriorated samples
Solution standards, even stored in
ampoules, are not reliable (esp.
when dissolved in ACN)
A UV-Vis detector set at 330nm may
be used
Iron contamination caused from
samples, long column storage,
stainless steel frits in the eluant
reservoirs, corrosion in system
Retention times not stable, especially

in early part of chromatogram
Re-equilibration time is too short.

Too much internal volume in HPLC
pump or pulse dampener.
Leaking proportioning valve in HPLC
Autosampler Problems
Increase it by two minute increments.

Re-plumb system as described in the
installation section for amino acids.
Artifacts in Baseline
Contamination in Eluant reservoir

Corrosion of spargers or filters
Volatile amines used in laboratory
Replace eluants
Clean reservoir with soap and water
Remove spargers or eluant filters
Remove any reagents used in amine
synthesis, or cigarette smoke laden
clothing
Retention times drift over a long time
Buildup of contaminants
Room temperature changes greatly
with the seasons
Flush the column

Air condition the room

7.6
Section 7
T roubleshooting
COMMON COLUMN PROBLEMS

OBSERVED PROBLEM
COMMON CAUSE
Loss of Resolution
Guard column dirty

Bad tubing connection: wrong style
nut, too large tubing, wrong type
union
Column worn out
Poor Peak Shape - General
Reagent backflows into column
High Column pressure
ACTION TO TAKE
NOTES
Column worn out

Guard column dirty
Deposits in post-column flow path
Partial obstruction of flowcell
Too strong a solvent
Too large a sample injected
Reagent flow rate too high.
Improper tubing connection.
Protein contamination on amino acid
column
Start by replacing filter, then guard.

Replace column as last resort.
If new tubing connections have been
made, check connections.
Ensure that protein is completely
removed form column
Send a chromatogram to Pickering

Laboratories Technical Support
Department
Improper tubing connections are:
wrong style nut, too large tubing,
wrong type union, improper swaging
of ferrule.
Reverse and flush ion-exchange
column at elevated temperature
Improper Shutdown procedures

Improper maintenance Procedures
Leaking fittings between column and
HPLC pump
Defective reagent control valves.
If NaOH is on column, follow

procedure on Page 7.9
Guard column is blocked.

Worn HPLC seal or rotor seal.
Particulate matter in eluant reservoirs
Column is damaged
Organic solvent in ion exchange
column
NaOH on Carbamate column
Excessive eluant flow rate through
column
For reagent back-flow onto column,

see above.
If the column back-pressure is high
(> 2800psi), isolate the source
of the high pressure and replace
appropriate part. The analytical
column can be back-flushed to clear
partial blockage. Disconnect the
outlet of the column during the back
flush operation.
If Reagent backflows onto column,

follow the procedure on Page 7.8
If organic solvent is on the cationexchange column, follow the

procedure on Page 7.8
.

Unfiltered samples
Pressure from filter and guard should
be < 200psi).
Organic contaminants can be washed
off the carbamate column by first
washing with methanol then with
dichloromethane. Wash again with
methanol before use
7.7
Section 7
T roubleshooting
APPLICATION SPECIFIC TROUBLESHOOTING

OBSERVED PROBLEM
COMMON CAUSE
ACTION TO TAKE
NOTES
Peak areas diminish
Old heated reactor
Replace heated reactor
Replace heated reactor every 2 years
Poor Aspartic Acid Shape
Wrong pH or buffer for sample
AMINO ACID
Threonine/Serine Resolution decreases Dirty guard

Worn Column
Reverse guard column and flush with

100% regenerant
Repack or replace guard/analytical
column
Hydrindantin Deposits in Reactor
Caused by out-of-date Trione
Clean entire system with ethanaol.

Replace reagent filter.
Fatty samples used in carbamate

analysis.
Follow the procedure on Page 7.11
Glyphosate peak is a doublet
Improperly buffered samples
Add 1-2 drops of Glyphosate RESTORE

to the sample
RESTORE Cat. No. 1700-0140
Glyphosate and AMPA peaks are late

and broad
Iron contamination of Column

Extremely large ID injection loop
Follow the procedure for pumping

RESTORE through the column
Replace the large ID loop with a

smaller ID.
CARBAMATES
Grease deposits in the heated reactor
GLYPHOSATE
Glyphosate peak too small or gone, but Oxidizing reagent too weak, too old,
AMPA present
NaOCl stock solution too old
AMPA peak disappears, but
Glyphosate present
Oxidizing reagent too strong
Make fresh Oxidizing reagent

7.8
Section 7
T roubleshooting
Software Troubleshooting
The software will alert you about common mistakes with Method and Sequence settings as well as with starting
up the Instrument. Read all the messages carefully.
Always collect the Log Files after problem is observed by going to Help Send log to support. It is essential
that log file be collected from the day the problem occurred. If you are not sure when exactly the problem
happened collect all 3 available logs.
Helpful tip: check the size of the log file after you save it. File that is less then 100 KB is likely to be empty or
corrupted. Delete it and collect the log again.
OBSERVED PROBLEM
COMMON CAUSE
ACTION TO TAKE
NOTES
Not Connected is displayed in the

status bar
Configuration is incorrect
Check that the correct connection

method is set in Configuration.
Contact your System Administrator for

IP address if needed.
Pinnacle refills full syringe instead of

just enough reagent for 1 run
Method not loaded
Create and load a Sequence with

correct post-column Method
After loading a new Sequence last

method from the old one is still
displayed
New settings did not get accepted
Restart the Pinnacle PCX software
Pinnacle does not refill after the first

run is completed
Pump was turned ON but Sequence

was not started
Injection signal was not received
Make sure Start is selected from the

Sequence menu
See below
After first run is started check that

Run time displayed in the Status
bar matches elapsed run time of
the HPLC.
No relay signal is displayed
Injection signal was not received and

Sequence was stopped
Check that correct brand of HPLC is set

in Configuration
Make sure that Equilibration time and
Run time of Pinnacle Method match
run and equilibration time of the
HPLC method (see section 4 of this
Manual)
Minimum Equilibration time for
Pinnacle PCX is 5 min
Check relay connections on the back of

your HPLC system and Pinnacle PCX.
Set the change of relay state in your
HPLC method close to time 0.0.
If equilibration time is set as part of
the gradient table consider how long
the actual analysis and equilibration
steps are. Match analysis time to
Run time of the Pinnacle PCX and
match time of the equilibration step
to Equilibration time of the Pinnacle
PCX.
continued

7.9
Section 7
T roubleshooting
SOFTWARE TROUBLESHOOTING
continued
OBSERVED PROBLEM
COMMON CAUSE
ACTION TO TAKE
NOTES
Pinnacle PCX misses command during

Sequence run (stops in the middle of
the Sequence, fails to refill, fails to
pump reagent, etc)
Slow communication between PC and

Pinnacle PCX
Collect the log file and contact

Pickering Laboratories to confirm the
cause of the problem.
If using USB cable change to Ethernet.
Make sure no updates are scheduled

on your computer when analysis is
running
Sequence done is displayed in

Pinnacle PCX software but not all
your samples were completed
Number of runs in Pinnacle PCX

Sequence and HPLC sequence do
not match
Pinnacle PCX sequence was modified
during the run but changes were
not saved
HPLC software usually displays only

More runs set up in HPLC sequence
than in Pinnacle PCX. Make sure you number of unknown samples and
do not count number of calibrators
match total number of runs in the
or control.
HPLC sequence.
Check number of runs displayed in the
When modifying currently running
status bar to confirm that changes
sequence press Save and then Load
were accepted
HIGH PRESSURE on Pump 1(2)
Pump pressure exceeded 500 psi
Completely or partially blocked:

reagent filters
heated or ambient reactors
detector flow cell
connecting tubings
backpressure regulator
See troubleshooting table for details on

dealing with blockages
Transducer 1(2) not connected
Transducer connection to the main

board is loose
Transducer or main board are
defective
Open the Pinnacle PCX right side

panel and check that transducer is
connected
Install a new transducer or a main
board
Contact Pickering Laboratories before

changing any major components
Unplug Pinnacle PCX before checking
electrical connections
Column temp sensor error
Column temperature sensor is not

connected to the main board
Column temperature sensor or main
board are defective
Open the Pinnacle PCX left side panel

and check that column temperature
sensor is connected
Install a new column temperature
sensor or a main board

continued

7.10
Section 7
T roubleshooting
SOFTWARE TROUBLESHOOTING
continued
Reactor temp sensor error
Heated reactor is not connected to the

main board
Heated reactor or main board are
defective
Open the Pinnacle PCX right side

panel and check that heated reactor
is connected
Install a new heated reactor or a
main board

Column temp at MAX
Column heater, column temperature

sensor or main board are defective
Send Pinnacle PCX to Pickering

Laboratories for repairs
Pinnacle PCX over temperature

switches are single use only. They
need to be replaced at the factory
before Instrument can be used again
Reactor temp at MAX
Reactor heater, reactor temperature

sensor or main board are defective
Send Pinnacle PCX to Pickering

Laboratories for repairs
Pinnacle PCX over temperature

switches are single use only. They
need to be replaced at the factory
before Instrument can be used again
Valve 1(2) lost
Valve can not find specified position
Valve is misaligned
Valves o-rings need replacement
Main board or valve motor and
sensors are defective
Buy valve resealing kit and change

o-rings. Align valve as described on
page 5-9
Replace main board or valve motor

7.11
Section 7
T roubleshooting
PROCEDURES
TO REMOVE SILICA DEPOSITS FROM REACTOR
Silica deposits are too hard to remove. Replace the reactor(s). Carefully clean or replace other components in
the flow path. You must remove all the silica before the system will work again. This will probably entail major
repair.
TO REMOVE MINERAL DEPOSITS IN THE REACTOR FROM HARD WATER
The Pickering pumps and most (but not all) HPLC pumps will tolerate this. Columns and autosamplers probably
will not tolerate this.
1. Remove analytical column
2. Start HPLC pump at < 0.5 mL/min (100% H2O).
3. Replace both post-column reagents with deionized water. Run post-column pumps for 510 min.
4. Stop post-column pumps. Replace deionized water with 20% nitric acid and run post-column pumps for
1015 min.
5. Reverse the order of washing with water and then replace with the post-column reagents.
TO REMOVE GREASE DEPOSITS
Grease deposits can be dissolved by replacing column and guard with a union and pumping methanol through
the HPLC and post-column systems. Stronger solvents such as acetone, methylene chloride, or tetrahydrofuran
(THF) may be needed. If methylene chloride is used, be certain to flush the system thoroughly with methanol
before and after because methylene chloride is not miscible with water.
IF REAGENT BACKFLOWS ONTO COLUMN
This procedure usually works but may not work every time.
1. Shut down the Pinnacle PCX.
2. Flush both columns with regenerant. Use a very slow flow rate so that the back pressure does not exceed
2000 psi. Collect effluent in a beaker.
3. Keep flushing until the pressure drops. Keep raising the flow rate until the pressure is normal at 0.40 mL/min
and 55C.
4. Run a chromatogram to check for resolution.
IF ORGANIC SOLVENT IS ON CATION-EXCHANGE COLUMN
Even small amounts of common organic solvents like Methanol or Acetonitrile will cause cation-excahnge resin
to swell leading to high column pressure. Always make sure all organic solvent are removed from HPLC system
and all the connecting lines before installing cation-exchange column. Small amounts of organic solvents from
HPLC system could be removed by the procedure below. Guard column and analytical column should be flushed
separately.


7.12
Section 7
T roubleshooting
1. Remove the column and flush HPLC system with water. Put column Regenerant on.
2. Connect the column in reversed direction and flush with Regenerant. Use a very slow flow rate so that the
back pressure does not exceed 2000 psi.
3. Keep flushing until pressure drops. Keep raising the flow rate until pressure is normal at standard operating
conditions. Normal pressure for guard column is < 500 psi. Normal column pressure without a guard <2000
psi.
4. Reinstall guard and analytical columns in normal direction.
5. Run a chromatogram to check for resolution.
IF NaOH IS ON COLUMN
1) Do not restart the system. Dissolved silica or C18 phase will reprecipitate in the post-column reactors, or
flowcell. These additional complications then require replacement of both reactor coils as well as your column.
2) Immediately depressurize the post-column system by loosening the To Detector fitting.
3) Disconnect the outlet of the column.
4) Restart the HPLC pump to flush the column with 100% MeOH for 20 minutes. Complete steps 24 as quickly
as possible because the longer the hydroxide stays inside the column, the less chance that the column will
survive.
5) Catch the effluent from the column with paper towels. Alternatively, connect the outlet of the column to a
piece of spare tubing directing the effluent to waste.
6) Turn off the HPLC pump and reconnect the outlet of the column and the To Detector fitting.
7) Turn on the HPLC and post-column system and run a calibration standard. Pay special attention to the first
four peaks. If these four peaks are not resolved, the column needs to be replaced.
TO REMOVE IRON CONTAMINATION FROM COLUMN
Flush guard and column with the Glyphosate Restore solution.
TO PUMP RESTORE THROUGH THE GLYPHOSATE COLUMNS
Usually only the guard column is contaminated. We suggest you buy a spare guard column to minimize
down-time.
1) Remove the analytical column after ensuring no residual post-column pressure.
2) Reverse the guard column and pump RESTORE through the guard at 0.4 mL/min for a minimum of 30 min,
directing the effluent to waste.
3) Pump K200 eluant through the guard long enough to displace RESTORE about 30 min
4) Reconnect the column and guard in the normal directions and restart the HPLC and post-column systems.
If analytical column is also contaminated, reverse the column and flush with Restore for 2 hrs. Equilibrate with
K200 for 1 hour before use.

8.1
A ppendices
Appendices
8.2
Installation/Operational Qualitication of the Pinnacle PCX

8.7
8.8 Performance Qualifications of the Pinnacle PCX Data Sheet
8.15
Sample Instrument Parameter Log
8.16 Flow Diagrams
Simplex
Duplex
8.18 Consumables and Spare Parts
Amino Acid Analysis
Carbamate Analysis
Glyphosate Analysis
8.24
Limited Warranty
8.25 References

8.2
A ppendices
Installation/Operational Qualitication of the Pinnacle PCX

Installation Qualification Completed By: ____________________________ Date: ______________
Operational Qualification Completed By: ___________________________ Date: ______________
1.0 PURPOSE:

To qualify the installation and operation of the Pinnacle PCX
2.0 REFERENCES:
2.1 Operators Manual for the Pinnacle PCX
2.2 Installation / Operational Qualification of the Pinnacle PCX Data Sheet. Section 8.3 of the Operators

Manual for the Pinnacle PCX.
3.0 EQUIPMENT:
3.1

3.2

Date Initials
Installation / Operation Qualification

Coordinator:
__________________________ _________ ___________
Equipment:
Asset #:
__________________________
Serial #:
__________________________
Location:
__________________________ _________ ___________
4.0 GENERAL INSTALLATION QUALIFICATIONS:

4.1

4.2
Date Initials
Pinnacle Site Requirements:
Space:
21.50 H x 10.63 W x 18.25 D inches (54.0 x 26.7 x 46.4 cm)
Electrical:
120 or 240 V
Weight:
26 lbs (11.6 kg) for Dual-pump systems
Gas:
Nitrogen or Helium
Pinnacle Installation Qualification:
4.2.1 Follow Section 3 in the Operators Manual for the Pinnacle PCX for installation details of

the Pinnacle PCX.
4.2.2 Complete the Installation Qualification Check List in the Installation Operation / Qualification of

the Pinnacle PCX Data Sheet, Section 8.7 of the Operators Manual for the Pinnacle PCX.
5.0 OPERATION QUALIFICATION:
Date Initials
5.1 Pump Flow Rate Accuracy

5.1.1 Turn on the Pinnacle PCX.
5.1.2 Fill the reagent bottle with Water and tightly close the cap.
5.1.3 Make sure the reagent caps are not vented to the atmosphere and that the lines to the pump are

open (parallel), and turn the gas toggle valve to ON.

8.3
A ppendices
5.1.4

5.1.5

5.1.6

5.1.7

Connect the outlet of Valve Port 2 directly to your flow meter. Use Valve 1 when testing Pump 1
and use Valve 2 when testing Pump 2.
Disconnect the analytical column to the Pinnacle PCX. This is the blue PEEK tubing that goes
into the first mixing tee. Replace the blue peek tubing with a plug into the mixing tee. This will
prevent any leaking out of the mixing tee. At this point the analytical column is completely
disconnected from the Pinnacle PCX.
Open the Pinnacle Control Software and configure the instrument to have no connection to
the HPLC. This will allow you to operate the Pinnacle without experiencing a time-out error due
to no injection signal from the HPLC.
Create a method with the following parameters:

8.4
A ppendices
5.1.8 Create a sequence to run the method in Step 5.1.7 once.

5.1.9

5.1.10
5.1.11

5.1.12

Save, and then load this sequence. Enable the instrument by selecting Enable under the
Control menu. Once the Pinnacle is enabled, refill the pumps by selecting Refill Pumps under the
Control menu.
Now start the sequence by selecting Start Sequence under the Sequence menu.
Allow a few minutes for the pump pressure to stabilize before recording a flow-rate reading. The
specifications for flow-rate accuracy are on the Installation / Operational Qualification of the
Pinnacle PCX Data Sheet, Section 8.8 of the Operators Manual for the Pinnacle PCX.
Repeat steps 5.1.7 through 5.1.11 for Pump 2. Make sure to program the flowrate of Pump 2 to
1.0ml/min and change the flowrate of Pump 1 to 0ml/min. If you have a single pump system,
proceed to the next step.
5.2 Column Heater Accuracy

5.2.1

5.2.2
Place a temperature probe or thermocouple inside of the column heater. Make sure the probe
or thermocouple is not touching the metal sides or the column door. The probe or
thermocouple must be measuring the temperature of the air inside the column heater.
Close the column heater door.

8.5
A ppendices
5.2.3 Create a method with the following parameters:

5.2.4 Create a sequence with one run of the method created in Step 5.2.3

5.2.5 Save and then Load the sequence that was created in Step 5.2.4.

8.6
A ppendices
5.2.6 Enable the instrument. This will cause the Pinnacle to heat the column to the temperature set in

the method created in Step 5.2.3
5.2.7 Allow enough time for the temperature to stabilize, and then record the temperature of the

column heater. The specifications for the column heater accuracy are in the Installation /

Operational Qualification of the Pinnacle PCX Data Sheet, Section 8.3 of the Operators Manual

for the Pinnacle PCX.

Does the equipment operate properly? YES / NO
5.3 Comments: ________________________________________________________________

_________________________________________________________________________
_________________________________________________________________________
_________________________________________________________________________
_________________________________________________________________________
_________________________________________________________________________
_________________________________________________________________________
6.0 MAINTENANCE PROCEDURES:
Date Initials
6.1 Are maintenance procedures (per Section 5 of the Operators Manual of the

Pinnacle PCX) scheduled as part of the Preventative Maintenance System?

YES / NO _________ ___________
6.2 Is an Equipment Activity Log available? YES / NO _________ ___________
7.0 INSTALLATION / QUALIFICATION ACCEPTANCE:

7.1 Criteria:
The equipment, as described in Section 3.2 of the Installation/Operational Qualifications of the Pinnacle
PCX, will be successfully installed and qualified if all equipment components are in place and operating
within the parameters and specifications originally set forth above. Any deviations or changes will be
noted with justification in the Section 5.3, Comments.
7.2 Approvals:
7.2.1 Installation/Operation Qualification:

These signatures verify that the equipment, as described in Section 4.2 of the Installation

Operational Qualifications of the Pinnacle PCX, meets the criteria for proper Installation

Operation Qualification.

Approved by (Quality): _________________________________ Date: _______________
Approved by (Management): _____________________________ Date: _______________

8.7
A ppendices

Serial Number:
120V or 240V
Customer:
Asset:
Installation Qualification Check List

Are all of the contents listed on the packing list present?
Yes / No
Is there enough bench space between the HPLC and detector so the Pinnacle Column Heater
Door and Pinnacle Cover can open without obstruction?
Yes / No
Is there at least 3 inches behind the Pinnacle PCX for ventilation?
Yes / No
Is there any damage on the outside of the Pinnacle PCX?
Yes / No
Is there any damage inside the Pinnacle PCX?
Yes / No
Are all electrical connections secure?
Yes / No
Is the gas source regulator set to a maximum of 75 psi?
Yes / No
Is a waste bottle provided by the user?
Yes / No
Is the HPLC correctly deadheaded? Refer to Section 3.9 or the Operators Manual for the
Pinnacle PCX.
Yes / No
Does the display turn on when the Pinnacle PCX is powered on?
Yes / No
Do the valves find home position when the Pinnacle PCX is turned on? The home position is when
the V on the valve is between 2 and 3.
Yes / No
Does the gas toggle valve turn the gas on and off?
Yes / No

8.8
A ppendices
Serial Number:
Customer:
120V or 240V
Asset:
Performance Qualifications of the Pinnacle PCX Data Sheet

Flow Rate Accuracy
Pump 1
Flowrate Setpoint in mL/min
1.0
Record Flowrate in ml/min
Specifications
+/- 0.030 ml/min
Pump 2
Flowrate Setpoint in mL/min
1.0
Record Flowrate in ml/min
Specifications
+/- 0.030 ml/min
Recorded Temperature in deg C
Specification
Pass / Fail
Pass / Fail
Column Heater Accuracy

Column Temperature Setpoint in deg C
45.0
+/- 1 deg C
Pass / Fail
Comments: ________________________________________________________________________
_________________________________________________________________________________
_________________________________________________________________________________
_________________________________________________________________________________
Performed By:__________________________________________________
Date Performed:______________
Reviewed By:___________________________________________________
Date Reviewed:_______________
Next Due Date:_________________________________________________

8.9
A ppendices
Serial Number:
Customer:
120V or 240V
Asset:
Glyphosate Application
Glyphosate Column Part Number:
Serial Number:
Glyphosate Guard column Part Number:

Glyphosate Test Mixture (p/n 1700-0080)
Lot Number:
Thiofluor (p/n 3700-2000)
Lot Number:
o-phthaladehyde (p/n O120)
Lot Number:
Glyphosate OPA Diluent (p/n GA104)
Lot Number:
Expiration Date:
Glyphosate Hypochlorite Diluent (p/n GA116)
Lot Number:
Expiration Date:
Glyphosate Potassium Eluant (p/n K200)
Lot Number:
Expiration Date:
Glyphosate Column Regenerant (p/n RG019)
Lot Number:
Expiration Date:
Glyphosate
Retention
Time in min
Average
Retention Time
Standard
Deviation
% CV
Run 1
Run 2
Run 3
Run 4
Run 5
Run 6
Glyphosate
Specification
Pass / Fail
0.5%
Peak Area
Average Peak
Area
Standard
Deviation
% CV
Run 1
Run 2
Run 3
Run 4
Run 5
Run 6
Specification
Pass / Fail
1.5%

8.10
A ppendices
Carbamate Application
Carbamate Column Part Number:
Serial Number:
Carbamate Guard column Part Number:

Carbamate Test Mixture (p/n 1700-0063)
Lot Number:
Thiofluor (p/n 3700-2000)
Lot Number:
o-phthaladehyde (p/n O120)
Lot Number:
Carbamate OPA Diluent (p/n CB910)
Lot Number:
Expiration Date:
Carbamate Hydrolysis Reagent (p/n CB130 or CB130.2) Lot Number:
Expiration Date:
Aldicarb
sulfoxide
Run 1
Run 2
Run 3
Run 4
Run 5
Run 6
Retention Time
Average
in min
Retention Time
Aldicarb
Retention Time
Average
in min
Retention Time
Standard
Deviation
% CV
Specification
Pass / Fail
0.5%
Standard
Deviation
% CV
Run 1
Run 2
Run 3
Run 4
Run 5
Run 6
Specification
Pass / Fail
0.5%
Aldicarb
sulfoxide
Run 1
Run 2
Run 3
Run 4
Run 5
Run 6
Peak Area
Aldicarb
Peak Area
Average
Peak Area
Standard
Deviation
% CV
Specification
Pass / Fail
1.5%
Run 1
Run 2
Run 3
Run 4
Run 5
Run 6
Average
Peak Area
Standard
Deviation
% CV
Specification
1.5%
Pass / Fail
8.11
A ppendices
Sodium Amino Acids Application
Amino Acid Column Part Number:
Serial Number:
Guard column Part Number:

Test Mixture (p/n 1700-0070)
Lot Number:
Trione (T100 or T200):
Lot Number:
Na Eluant Buffer A:
Lot Number:
Expiration Date:
Na Column Reagent (p/n RG011)
Lot Number:
Expiration Date:
Threonine
Retention Time
Average
in min
Retention Time
Standard
Deviation
% CV
Run 1
Run 2
Run 3
Run 4
Run 5
Run 6
Serine
Pass / Fail
0.5%
Retention Time
Average
in min
Retention Time
Standard
Deviation
% CV
Run 1
Run 2
Run 3
Run 4
Run 5
Run 6
Cysteic Acid
Specification
Specification
Pass / Fail
0.5%
Retention Time
Average
in min
Retention Time
Standard
Deviation
% CV
Run 1
Run 2
Run 3
Run 4
Run 5
Run 6
Specification
Pass / Fail
0.5%

8.12
A ppendices
Threonine
Peak Area
Average Peak
Area
Standard
Deviation
% CV
Run 1
Run 2
Run 3
Run 4
Run 5
Run 6
Serine
Pass / Fail
1.5%
Peak Area
Average Peak
Area
Standard
Deviation
% CV
Run 1
Run 2
Run 3
Run 4
Run 5
Run 6
Cysteic Acid
Specification
Specification
Pass / Fail
1.5%
Peak Area
Average Peak
Area
Standard
Deviation
% CV
Run 1
Run 2
Run 3
Run 4
Run 5
Run 6
Specification
1.5%
HPLC Method Table

Time
0
20
20.1
24
24.1
Stop Time
Post Time
Buffer A*
100
100
0
0
100
RG011
0
0
100
100
0
24 min
12 min
* Use the A Buffer corresponding to the column you have. The column insert will have
the correct initial buffer listed in the method.

Pass / Fail
8.13
A ppendices
Lithium Amino Acids Application
Amino Acid Column Part Number:
Serial Number:
Guard column Part Number:

Test Mixture (p/n 1700-0070)
Lot Number:
Trione (T100 or T200):
Lot Number:
Li Eluant Buffer A:
Lot Number:
Expiration Date:
Li Column Reagent (p/n RG003)
Lot Number:
Expiration Date:
Threonine
Retention Time
in min
Average
Retention Time
Standard
Deviation
% CV
Run 1
Run 2
Run 3
Run 4
Run 5
Run 6
Serine
Pass / Fail
0.5%
Retention Time
in min
Average
Retention Time
Standard
Deviation
% CV
Run 1
Run 2
Run 3
Run 4
Run 5
Run 6
Cysteic Acid
Specification
Specification
Pass / Fail
0.5%
Retention Time
in min
Average
Retention Time
Standard
Deviation
% CV
Run 1
Run 2
Run 3
Run 4
Run 5
Run 6
Specification
Pass / Fail
0.5%

8.14
A ppendices
Threonine
Peak Area
Average Peak
Area
Standard
Deviation
% CV
Run 1
Run 2
Run 3
Run 4
Run 5
Run 6
Serine
Pass / Fail
1.5%
Peak Area
Average Peak
Area
Standard
Deviation
% CV
Run 1
Run 2
Run 3
Run 4
Run 5
Run 6
Cysteic Acid
Specification
Specification
Pass / Fail
1.5%
Peak Area
Average Peak
Area
Standard
Deviation
Run 1
Run 2
Run 3
Run 4
Run 5
Run 6
% CV
Specification
1.5%
Method Table
Time
0
20
20.1
24
24.1
Stop Time
Post Time
Buffer A*
100
100
0
0
100
RG003
0
0
100
100
0
Column Temp: Refer to column insert.

Eluant Flow Rate: Refer to column insert.
Reactor Temperature: 130 deg C
Reagent Flow Rate: 0.3mL/min
24 min
12 min
* Use the A Buffer corresponding to the column you have. The column insert will have
the correct initial buffer listed in the method.

Pass / Fail
8.15
A ppendices
Sample Instrument Parameter Log

NORMAL PRESSURES AND FLOWS AT INITIAL CONDITIONS
Flow Rate
Pressure
Set Temperature
Actual Temperature
Analytical Pump (Column)

Reagent Pump 1
Reagent Pump 2
TEMPERATURE
Analytical Column
Heated Reactor
Room

8.16
A ppendices
R1
FLUSH
Flow Diagram Simplex

Nut: 1452-0118
Ferrule: 1452-0117
Nut: 1452-0180
Ferrule: 1452-0181
1452-0182
Nut: 1452-0118
Ferrule: 1452-0117
1452-0040
2103-0463
COLUMN
FILTER 3102-9040
2102-0463
HEATED
REACTOR
TRANSDUCER
1452-0187
1552-0017
1452-0190
Nut: 1452-0113
Ferrule: 1452-0115
Nut: 1452-0114
Ferrule: 1452-0112
2
1452-0184
Coupler: 3102-3064
Nut: 1452-0113
Ferrule: 1452-0115
1452-0093
3
Nut: 1452-0116
Ferrule: 1452-0115
PUMP
VALVE 1 5
4
2102-0463
Nut: 1452-0118
Ferrule: 1452-0117
Nut: 3101-0011
Ferrule: 1452-0112
1452-0185
Check Valve: 3106-1007

Nut: 1452-0226
Ferrule: 1452-0112
WASTE
TO DETECTOR
FROM
HPLC
bold = kit

Nut: 1452-0118
Ferrule: 1452-0117
Union: 3102-3063
Back Pressure
Regulator: 3102-9025
8.17
A ppendices
Flow Diagram Duplex
1552-0017
Nut: 1452-0180
Ferrule: 1452-0181
1452-0184
COLUMN
1452-0189
Nut: 1452-0116
Ferrule: 1452-0115
R1
R2
TRANSDUCER 2
Nut: 1452-0118
Ferrule: 1452-0117
FLUSH
Nut: 1452-0113
Ferrule: 1452-0115
PUMP 2
1452-0188
2102-0463
1452-0093
1452-0185
3102-9040
3 VALVE 2 5
4
2103-0463
FILTER
1452-0041
1452-0183
Nut: 1452-0118
Ferrule: 1452-0117
1100-2927
AMBIENT
REACTOR
HEATED
REACTOR
Nut: 1452-0113
Ferrule: 1452-0115
Nut: 1452-0118
Ferrule: 1452-0117
TRANSDUCER 1
1552-0017
1452-0040
1452-0188
Nut: 1452-0114
Ferrule: 1452-0112
1452-0189
3102-9040
Coupler: 3102-3064
1452-0184
FILTER
PUMP 1
2102-0463
Nut: 1452-0113
Ferrule: 1452-0115
1452-0093
3
VALVE 1 5
4
Nut: 1452-0118
Ferrule: 1452-0117
Nut: 3101-0011
Ferrule: 1452-0112
WASTE
TO DETECTOR
Check Valve: 3106-1007

Nut: 1452-0226
Ferrule: 1452-0112
Nut: 1452-0118
Ferrule: 1452-0117
Union: 3102-3063
Back Pressure
Regulator: 3102-9025
1452-0185
FROM
HPLC
bold = kit
Nut: 1452-0116
Ferrule: 1452-0115

8.18
A ppendices
Parts List for Pinnacle PCX

CATALOG NUMBER
DESCRIPTION
Reservoirs and Caps

3107-0137
Rservoir Bottle, safety-coated, 1L
3107-0147
Cap Assembly for 1L bottle, includes cap with integrated valve
3107-0300
Reservoir Assembly, includes 1L bottle, and cap with integrated valve
1452-0120
Bottle, Piston Wash with Cap
1452-0121
Bottle, System Flush with Cap
3107-0149
Bottle, 1L, clear
1925-0129
Cap for Flush Bottle GL - 38
1925-0130
Cap for Wash bottle GL - 38
Tubing
2101-0212
PTFE tubing, 1/16OD x 0.01ID, 3ft
2101-0225
PTFE tubing, 1/16OD x 0.025ID, 3ft
2103-0463
Air Barrier Tubing, 1/8 OD x 1/16 ID
2104-0210
Inert PEEK tubing, 1/16OD x 0.01ID, 3ft
2104-0220
Inert PEEK tubing, 1/16OD x 0.02ID, 3ft
2101-0232
C-flex tubing, 1/4OD x 1/8ID, 3ft
2102-0463
FEP (clear) Tubing, 1/8OD x 0.063ID, 3ft
1452-0182
Tubing Assembly, Column out-Manifold, Single Pump System
1452-0183
Tubing Assembly, Column out-Manifold, Dual Pump System
1452-0184
Tubing Assembly, Pump-Transducer (2ea)
1452-0185
Tubing Assembly, Valve-Pump (2ea)
1452-0186
Tubing Assembly, Gas Manifold-Bottle (2ea)
1452-0187
Tubing Assembly, Valve-Manifold, Single Pump System
1452-0188
Tubing Assembly, Transducer-Valve, Dual Pump
1452-0189
Tubing Assembly, Valve-Manifold, Dual Pump System
1452-0190
Tubing Assembly, Transducer-Valve, Single Pump
1100-0450
Tubing Assembly, Pinnacle PCX to HPLC
1100-0460
Dead Head Plumbing Kit, Pinnacle PCX to HPLC
1100-0500
Tubing Kit, Dual Pump Pinnacle
1100-0501
Tubing Kit, Single Pump Pinnacle

8.19
A ppendices
Nuts and Ferrules

3101-0011
Nut Female, 1/4-28, PEEK
1452-0112
Ferrule, Short, PEEK, 1/16, 1/4-28, pack of 5
1452-0113
Nut, Short, PEEK, 1/8, 1/4-28, pack of 5
1452-0114
Nut, Short, PEEK, 1/16, 1/4-28, pack of 5
1452-0115
Ferrule, PEEK, 1/8, 1/4-28, pack of 5
1452-0116
Nut, PEEK, 1/8, 1/4-28, pack of 5
1452-0117
Ferrule, 1/16, Lite Touch, pack of 5
1452-0118
Nut, 1/16, Lite Touch, pack of 5
1452-0180
Nut, 1/4-28, 1/8, Polypropylene, pack of 5
1452-0181
Ferrule, 1/4-28, 1/8, Tefzel, pack of 5
1452-0226
Nut, PEEK, 1/8, 1/4-28 High Pressure, pack of 5
Pump Parts
1352-0007
Pump Cylinder, Ceramic, 70mL
1452-0038
Pump Assembly, 70mL with motor
1452-0122
Kit, Pinnacle Pump Seal
Valve Parts
1452-0045
Valve Assembley, Valve face and motor
1452-0093
Valve face Assembley, Liquid end
1452-0201
Kit, Valve seals
1452-0202
Kit Pinnacle Valve Maintenance
Reactor Cartridge Coils & Knitted

1452-0094
Reactor Cartridge Coil Assembly - 0.15mL 130C
1452-0064
1452-0095
1452-0096
1452-0097
Reactor Cartridge Knitted Assembly - 2.0mL 130C
1452-0098
1452-0099
1452-0100
Reactor Cartridge Knitted Assembly - 1.2mL & 1.6mL 130C

8.20
A ppendices
Miscellaneous
1100-2927
Ambient Reaction Coil
1352-0055
Drip Tray
1452-0040
Liquid Manifold with relief valve assembly
1452-0041
Liquid T Manifold assembley
1452-0064
Reactor Cartridge Coil Assembly - 0.5mL
1452-0141
Gas Manifold Assembley
1552-0017
Pressure Transducer Assembly
3101-0020
Plug, 1/4-28, Deldrin
3101-0030
Lpug, 10-32, 1/16, Deldrin
3102-3063
Union, PEEK ZDV
3102-3064
One Piece Coupler
3102-9025
Back Pressure Regulator, 100 psi
3102-9161
Tubing Cutter for 1/8-1/16 tubing
3106-1007
Inline Check Valve
3551-0073
Fuse, 5A Time lag high break ceramic
3560-2000
Cable, RS232
1452-0126
Relay Cable Assembly
3102-9040
Frit Replacement PEEK, 10m
1452-0327
USB Cable

8.21
A ppendices
Recommended Consumables and Spare Parts

For routine maintenance and minimal interruptions to your operation, always keep the necessary consumables
and spare parts available.
AMINO ACID ANALYSIS
Post-Column Reagents
CATALOG NUMBER
DESCRIPTION
O120
o-Phthalaldehyde (OPA), Chromatographic Grade crystals, 5g
OD104
OPA Diluent for Amino Acid Analysis, case of 4 ( 950mL per bottle)
3700-2000
Thiofluor, Chromatographic Grade crystals, 10g
T100
TRIONE Ninhydrin Reagent, 3-month* shelf life, 950 mL
T100C
TRIONE Ninhydrin Reagent, 3-month* shelf life, case of 4 (950 mL per bottle)
T200
TRIONE Two-part Ninhydrin Reagent (12 month* shelf life before mixing),
prepares 4 x 950 mL
* From date of manufacture
Sodium Columns and Eluants

CATALOG NUMBER
DESCRIPTION
Columns
1154150T
High-efficiency sodium ion-echange column, 4.0 x 150 mm, protein, collagen, and
oxidized hydrolysate
1154150
High-efficiency sodium ion-exchange column, 4.0 x 150 mm, oxidized hydrolysate
1154110T
High efficiency sodium ion-exchange column, 4.6 x 110 mm
1193250
Sodium ion-exchange column, 3.0 x 250 mm
1700-3100
GARD Holder
1700-3101
Cation GARD (2 ea)
1700-3101
Kit, GARD Holder and two Cation GARDs
Eluants
Na270
Sodium eluant, pH 2.80, case of 4 ( 950mL per bottle)
1700-0112*
Sodium eluant, pH 3.15, 5% sulfolane, case of 4 ( 950mL per bottle)
Na328
Na740
RG011
Sodium column regenerant (950mL)
Na220
Sodium sample diluent, pH 2.20 case of 4 ( 250mL per bottle)
1700-0155
Calibration standard, oxidized feed hydrolysate, 0.25 mole/mL, (5mL)

Continued on next page

8.22
A ppendices
Calibration Standards
012506H
Calibration standard, protein hydrolysate, 0.25 mole/mL, (5mL)
012506C
Calibration standard, collagen hydrolysate, 0.25 mole/mL, (5mL)
1700-0070
Amino acid test mixture, 3-component, 0.25 mmol/mL (1.5mL)
* For 1154150 columns with serial numbers after 1314
Lithium Columns and Eluants

CATALOG NUMBER
DESCRIPTION
Columns
0354675T
High-efficiency lithium ion-exchange column, 4.6 x 75 mm
0354100T
High-efficiency lithium ion-exchange column, 4.0 x 100 mm
0393250
Lithium ion-exchange column, 3.0 x 250 mm
1700-3100
GARD Holder
1700-3101
Cation GARD (2 ea)
1700-3101
Eluants
1700-1125
Lithium eluent pH 2.80 case of 4 (950mL per bottle)
Li220
Lithium sample diluent pH 2.36 (950mL)
Li275
Lithium eluent pH 2.75 case of 4 ( 950mL per bottle)
Li280
Li292
Li365
Li375
Li750
RG003
Lithium column regenerant (950mL)
Calibration Standards
011006P
Calibration standard, with norleucine, 0.25 mole/mL (5mL)
012006P
standard without norleucine, 0.25 mole/mL (5mL)
1700-0070
Amino acid test mixture, 3-component, 0.25 mole/mL (1.5mL)
1700-0180
Native Sample Standard, acidics and neutrals, in 0.1 N HCl, 5 mL
1700-0175
Native Sample Standard, basics, in 0.1 N HCl, 5 mL
1700-0170
Native Sample Standard without Norleucine & Alpha-Amino-Betaguanidinopropionic acid, in 0.2 N Lithium citrate buffer pH 2.36, 5 mL
Sample Preparation
SP 100
SERAPREP, for sample preparation of serum, (250mL)
UP 100
URIPREP, for sample preparation of urine (250mL)

8.23
A ppendices
CARBAMATE ANALYSIS
Reagents
CATALOG NUMBER
DESCRIPTION
O120
o-Phthalaldehyde, Chromatographic Grade crystals, 5 g
3700-2000
Thiofluor, Chromatographic Grade crystals, 10 g
CB910
OPA Diluent for Carbamate Pesticide Analysis, 4 x 950 mL
CB130
Hydrolysis Reagent for Carbamate Pesticide Analysis, 4 x 950 mL
CB130.2
Hydrolysis Reagent for EPA 531.2, 4 x 950mL
1700-0063
Carbamate Test Mixture, qualitative sample, 12 components, 1.5 mL,2.5 g/mL
1700-0132
ChlorAC Buffer for preservation of aqueous carbamate samples, 250 mL
Columns & Guards

CATALOG NUMBER
DESCRIPTION
0840250
Carbamate C8 column, 4.0 mm ID x 250 mm (with carbamate test mixture 1700-0063)
1846150
0846250
18ECG002
Replacement Carbamate Guard Cartridges - (Qty. 2)
18ECG001
Guard Cartridge holder with 3 guard cartridges
GLYPHOSATE ANALYSIS
Reagents
CATALOG NUMBER
DESCRIPTION
O120
o-Phthalaldehyde, Chromatographic Grade crystals, 5 g
3700-2000
Thiofluor, Chromatographic Grade crystals, 10 g
GA104
OPA Diluent for glyphosate analysis, 4 x 950 mL
GA116
Hypochlorite Diluent for glyphosate analysis, 4 x 950 mL
K200
Eluent for glyphosate analysis, 4 x 950 mL
RG019
Column Regenerant for glyphosate analysis, 4 x 950 mL
1700-0080
Test mixture, 2.5 g/mL each glyphosate and AMPA, 1.5 mL
1700-0140
RESTORE for removal of metal ion contamination from glyphosate column and guard, 250 mL
Columns & Guards

CATALOG NUMBER
DESCRIPTION
1954150
Glyphosate column, 4.0 mm ID x 150 mm (with Glyphosate test mixture1700-00??)
1700-3100
GARD Holder
1700-3101
Cation GARD (2 ea)
1700-3101
1705-0001
Sample clean up cartridge, SPE column caution exchange (10pkg)


8.24
A ppendices
Limited Warranty
INSTRUMENTS
Pickering Laboratories, Inc., (Pickering) Instruments are warranted to be free of defects in
material and workmanship under normal installation, use, and maintenance, for a period
of one year from the date of delivery to the Customer. Pickering will replace or repair,
without cost, any defective items. Expendable items such as check valves, pistons, piston
seals, and filters are excluded from this warranty. In addition, physical damage, poor quality
reagent- and sample-induced damage, and instrument damage due to Customers
misuse are not covered by this warranty.
ANALYTICAL COLUMNS
Pickerings Analytical Columns are warranted to be free of defects in materials and
workmanship under normal installation, use, and maintenance, for the warranted time
beginning from the date of delivery to the original Customer. Pickering will replace the
Analytical Column under warranty if found defective in material or workmanship. However,
the warranty is void if the Analytical Column was damaged due to Customers misuse.
Columns are warranted for 90 days.
HOW TO OBTAIN WARRANTY SERVICE
If there is a problem with your Instrument or Analytical Column within the Warranty
period, do not attempt to repair. Immediately notify Pickering at (800) 654-3330; if
calling from outside U.S.A., use (650) 694-6700. If the Instrument or Analytical Column
was not purchased directly from Pickering, please contact the vendor where it was
purchased. Any Instrument, part of the Instrument, or Analytical Column returned to
Pickering for examination or repair shall have Pickerings prior approval (call for a
Returned Goods Authorization number) and be sent prepaid by the Customer. Return
transportation will be at Pickerings expense if the Instrument, part of the Instrument, or
Analytical Column is found to be defective and under warranty.
Pickering Laboratories, Inc.

1280 Space Park Way
Mountain View, CA 94043
U.S.A.

8.25
A ppendices
References
INSTRUMENTATION
M.V. Pickering, Assembling an HPLC post-column system: practical considerations,
LCGC, 6, 11 (1988) 994997.*
M.V. Pickering, Modifying HPLC equipment to tolerate corrosive solutions, LCGC, 6, 9 (1988) 800809.*
J.W. Dolan and L.R. Snyder, Troubleshooting LC Systems, Humana Press, Clifton, NJ (1989).
* Reprints available from Pickering Laboratories

AMINO ACID ANALYSIS
D.H. Spackman, W.H. Stein and S. Moore, Anal. Chem., 30 (1958) 1190.
M. V. Pickering, LCGC, 7 (1988) 484.*
J. A. Grunau and J.M. Swiader, J. Chromatogr., 594 (1992) 165.*
A. A. Boulton, G. B. Baker and J.D. Wood (Eds.), Neuromethods 3, Amino Acids,
Humana Press, Clifton, NJ (1985), Chapter 1.
CARBAMATES ANALYSIS
Measurement of N-methyl carbamoyloximes and N-methyl carbamates in drinking water by direct aqueous
injection LC with post-column derivatization, EPA Method 531 by D.L. Foerst, EPA/600/4-851054 (1986);
Method 5, revised by T. Engels, National Pesticide Survey, Battelle Columbus Lab (1987); Method 531.1,
revised by R.L. Graves, EPA, Environmental Monitoring and Support Laboratory, Cincinnati (1989).
M. W. Dong, F.L. Vandemark, W.M. Reuter, and M.V. Pickering, Carbamate pesticides analysis by liquid
chromatography, Amer. Environ. Lab., 2(3) (1991) 1427.
K.M. Hill, R.H. Hollowell, and L. D. Dal Cortivo, Determination of N-methylcarbamate pesticides in well
water by liquid chromatography with post-column fluorescence derivatization, Anal. Chem., 56 (1984)
24652475.
H. Frister, H. Meisel, and E. Schlimme, OPA Method Modified by Use of N, N-Dimethyl 2-mercaptoethylammonium Chloride, Fresenius Z. Anal. Chem., 330 (1988) 631633.
GLYPHOSATE ANALYSIS
J.E. Cowell, Analytical Residue Method for N-Phosphono-methylglycine and
Aminomethylphosponic acid in Environmental Water, Monsanto Method Number 86-63-1, 1987
Environmental Protection Agency Draft Method 597: Analysis of Glyphosate in Drinking Water by Direct
Aqueous Injection LC with Post-Column Derivatization.

Index
A
Air Barrier tubing 2.6, 2.9, 2.14, 3.2, 3.5, 3.6, 3.12, 8.18
Ambient Reactor 2.7, 3.11, 5.7, 5.8, 8.3
Amino Acid 6, 7, 3.1, 3.3, 3.9, 3.14, 3.15, 6.2-5, 6.2-18, 8.11, 8.13, 8.21, 8.25
AMPA 6.4, 6.4-1, 6.4-2, 6.4-3, 6.4-5, 6.4-6, 8.23
Analytical Method 4.14
AOAC 6.2-2, 6.3, 6.3-1, 6.3-2, 6.4-1
Aqueous Sample 6.3-6, 6.3-7, 6.3-9, 6.3-11, 6.3-12, 6.4-7
Automatic Shutdown 3.15, 4.18, 5.4
Autosampler 6, 3.3, 3.8, 6.2, 6.3
Back-flow 2.13, 5.3, 6.3-12, 6.4-6

Background Signal 2
Back Pressure Regulator 3.11, 5.2, 6.2-19, 8.20
Buffer 7, 3.10, 3.11, 5.2, 5.3, 5.6, 6.2-3, 6.2-20, 6.3-3, 6.3-12, 6.4-7, 8.11, 8.12, 8.13, 8.14,
8.22, 8.23
Calcium Hypochlorite 6.4-3

Calibration 6, 6.3-12, 6.4-7, 8.21, 8.22
Cap 2.6, 3.12, 3.13, 5.9, 5.11, 5.13, 5.14, 5.15, 6.2-3, 6.2-4, 6.3-4, 6.4-3, 8.18
Carbamate 6, 3.1, 3.4, 3.10, 3.15, 6.3, 6.3-1, 6.3-2, 6.3-3, 6.3-5, 6.3-6, 6.3-7, 6.3-8, 6.3-9, 6.3-10,
6.3-11, 6.3-12, 8.10, 8.25
Check Valve 2.3, 2.13, 3.9, 3.10, 5.3, 8.20
Check Valves 8.24
Chemistry 6, 3.4
ChlorAC 6.3-3, 8.23
Collagen Hydrolysate 6.2-13, 6.2-15, 6.2-16, 8.22
Column and Guard Installation 3.10
Column Connections 2.3, 3.2
Column Oven 2.3, 2.4, 2.11, 3.5, 3.10, 3.11, 4.2, 4.8, 5.6, 6.2-5, 6.2-6, 6.2-8, 6.2-11, 6.2-12, 6.2-13
Column Pressure 5.3, 6.2-18, 6.4-4, 7.6, 7.11, 7.12
Column Temperature 4.6, 4.8, 6.2-6, 6.2-8, 6.2-11, 6.2-20, 6.3-6, 6.3-8, 6.3-10, 6.3-11, 6.4-4,
7.9, 7.10, 8.8
Computer Connections 3.6
Computer Requirements 3.4, 4.1
Configure 3.6, 3.7, 4.6, 8.3
Consumables 8.21
Contaminants 3.16, 4.18, 5.3, 5.4, 6.2-19, 6.3-4, 6.3-12, 6.4-3, 6.4-6, 7.5, 7.6
Contamination 2.5, 2.6, 3.8, 3.12, 3.13, 5.3, 6.2-18, 6.2-19, 6.4-1, 6.4-6, 7.4, 7.5, 7.6, 7.7, 8.23
Index
D
Dead-Head 6, 3.9
Deposits in Reactor 7.7
Designing a Post-column System 4
Detector Connections 2.8, 3.11
Detector Settings 4.14
Display Module 2.10
Electronics Compartment 2.11

Eluant 6, 2, 2.3, 3.1, 3.2, 3.10, 3.14, 3.15, 3.16, 4.18, 5.2, 5.3, 5.4, 5.5, 5.6, 6.2, 6.2-18, 6.2-19,
6.4-1, 6.4-7, 7.3, 7.4, 7.5, 7.6, 7.12, 8.9, 8.11, 8.13, 8.14, 8.21, 8.22
Empty Pump 3.13, 4.4, 4.15, 5.9
Enable 3.14, 4.3, 4.4, 4.5, 4.15, 5.5, 8.4, 8.6
EPA 5.3, 6.3-2, 6.3-3, 6.3-12, 6.4-3, 6.4-7, 8.23, 8.25
Equilibration 3.14, 4.8, 4.9, 4.10, 4.12, 4.16, 4.18, 6.2-7, 6.2-8, 6.2-9, 6.2-10, 6.2-16, 6.2-17,
6.2-18, 6.4-4, 6.4-6, 6.4-7, 7.5, 7.8
Error Messages 4.3, 4.7, 7.2, 7.9, 7.10
Ethernet 3, 5, 2.11, 2.12, 3.7, 4.1, 7.9
Ferrules 2.6, 3.2, 3.5, 5.1, 5.7, 8.19

Fittings 2.13, 3.2, 3.11, 5.2, 5.3, 5.8, 5.9, 6.2-18, 7.3, 7.6
Flow Path 2.2, 3.3, 7.3, 7.6, 7.11
Fluidics Panel 2.6, 2.7, 3.5, 3.11, 5.6, 5.8
Flush Method 4.9, 4.11, 4.17, 5.5
Flush Pump 3.13, 4.4, 5.9, 5.12, 5.16, 7.3
Flush Reservoir 2.7, 3.12, 3.13
Fuse 3, 2.11, 2.12, 5.2, 5.16, 8.20
Gas Connections 3.5, 3.6

Gas Manifold 2.6, 2.9, 2.14, 8.18, 8.20
Glyphosate 5, 6, 3.1, 3.3, 3.10, 3.15, 6.4, 6.4-1, 6.4-2, 6.4-3, 6.4-4, 6.4-5, 6.4-6, 6.4-7, 7.4, 7.5,
7.7, 7.12, 8.9, 8.23, 8.25
Grease Deposits 7.7, 7.11
Index
H
Heated Reactor 2, 2.7, 2.9, 2.13, 2.14, 3.11, 3.15, 4.2, 4.17, 5.2, 5.3, 5.5, 5.6, 5.8, 6.2-19, 7.4,
7.7, 7.10, 8.3, 8.15
Help 2, 4, 6, 2.5, 4.5, 4.7, 4.9, 4.11, 5.2, 7.2, 7.4, 7.8
High Pressure 5.3, 6.2-18, 7.6, 7.9, 8.19
HPLC Slowdown method 3.16, 4.18, 5.4
HPLC Synchronization 4.1
HPLC System Requirements 3.3
Hydrindantin 6.2-1, 7.4, 7.7
Hydrolysis Reagent 6, 6.3-3, 6.3-4, 6.3-5, 7.5, 8.10, 8.23
Important Practical Considerations 4.9, 4.16

Installation 1, 3, 2.10, 3.2, 3.4, 3.6, 3.10, 4.3, 4.6, 4.15, 5.1, 6.3-3, 7.5, 8.2, 8.4, 8.6, 8.7, 8.24
Installation Qualification 8.2, 8.7
Ion-exchange 3.16, 4.18, 5.3, 5.4, 6.2, 6.2-1, 6.2-18, 6.3-12, 7.6, 8.21, 8.22
Ion-exclusion 6.2-1
Iron Contamination 6.4-1, 6.4-6, 7.5, 7.7, 7.12
LCD 3, 2.10, 2.14, 3.6, 3.7, 4.3

Leaks 3.6, 3.10, 5.2, 5.7, 5.8, 6.2-18, 6.2-19, 7.3
Lithium ion-exchange 6.2, 8.22
Log Files 4.7, 7.2, 7.8
Long-Term Shutdown 5.4, 5.5
Maintenance 1, 4.5, 5.2, 5.5, 5.6, 5.9, 5.10, 5.12, 7.2, 7.6, 8.6, 8.19, 8.21, 8.24
Manual Shutdown 3.15, 4.17, 5.4
Methanolic Samples 6.3-8, 6.3-10, 6.3-12
Mineral Deposits 7.4, 7.11
Mixing Manifold 2.7, 5.6, 5.8
MODE button 3.7, 3.8
MSDS 3.1, 5.2
MSUD 6.2-10
Network 3, 5, 2.10, 2.12, 3.4, 3.6, 3.7, 4.1, 4.6

Ninhydrin 7, 3.2, 6.2, 6.2-1, 6.2-2, 6.2-4, 6.2-20, 8.21
Noisy Baseline 7.4
Nuts 2.6, 3.2, 3.5, 5.1, 5.7, 8.19
Index
O
OPA 6, 7, 3.2, 4.12, 5.3, 6.2, 6.2-2, 6.2-4, 6.2-5, 6.2-19, 6.3, 6.3-2, 6.3-4, 6.3-5, 6.3-12, 6.4-1,
6.4-3, 6.4-6, 7.5, 8.9, 8.10, 8.21, 8.23, 8.25
Operational Qualification 8.2, 8.4, 8.6
Organic Solvents 3.10, 6.2-18, 6.3-12, 6.4-7, 7.11
O-ring 2.4, 5.10, 5.14, 5.15, 7.10
Over-Pressure Relief 2.7, 2.13, 5.6, 7.4
Oxidized Feed Hydrolysate 8.21
Oxidizing Reagent 6.4-2, 6.4-3, 7.7
Parameter Log 5.1, 7.2, 7.3, 8.15

Parts List 3.10, 8.18
Pause Sequence 4.5
Peak Identification 6.2-6, 6.2-7, 6.2-8, 6.2-9, 6.2-11, 6.2-14, 6.2-15, 6.2-17
Physiological Fluid 6.2-2, 6.2-6, 6.2-7, 6.2-9
Piston Head 5.13, 5.14, 5.15
PKU 6.2-10
Post Column Conditions 6.2-4
Post-Column Derivatization 2, 1, 3.1, 6.2, 6.3, 6.4, 8.25
Precautions 6.2, 6.2-18, 6.3, 6.3-12, 6.4, 6.4-6
Precipitate 2, 5.2, 5.3, 6.2-19, 6.3-3, 6.3-4, 6.3-12, 6.4-3, 6.4-6
Pre-Column Check Valve 3.9, 3.10, 5.3
Pressure Transducer 6, 2.7, 5.6, 5.7, 8.20
Primary Amines 6.2-1, 6.2-2, 6.2-19
Priming 3.10
Protein Hydrolysate 6.2, 6.2-2, 6.2-12, 6.2-16, 6.2-17, 8.22
Pump Flush 5.11
Pump Parts 8.19
Pump Seal 5.2, 5.12, 5.14, 7.3, 7.4, 8.19
Reactor Temperature 3.15, 4.2, 4.6, 4.7, 4.8, 4.18, 5.4, 7.2, 7.5, 7.10, 8.14
Reagent Backflow 7.6, 7.11
Reagent Filter 2.7, 3.2, 5.2, 5.6, 5.7, 7.7, 7.9
Reagent Preparation 6, 7, 6.2-3, 6.3-3, 6.4-2, 7.5
Reagent Pressure 5.2, 7.3
Reagent Pump 2, 1, 2, 2.4, 2.9, 2.14, 3.2, 3.13, 3.14, 4.2, 4.6, 4.9, 4.10, 7.3, 7.5, 8.15
Reagent Reservoir 2, 5, 2.5, 2.7, 2.9, 2.14, 3.2, 3.12, 6.2-3, 6.2-4, 6.3-5, 6.4-2, 6.4-3
Reagent Stability 2
Reagent Valve 2.5
Index
R
Regenerant 3.3, 3.16, 4.18, 5.4, 6.2-18, 6.2-20, 7.5, 7.7, 7.11, 7.12, 8.9, 8.21, 8.22, 8.23
Relay 3, 5, 2.11, 2.12, 3.4, 3.6, 3.14, 4.1, 4.2, 4.3, 4.6, 4.14, 4.15, 7.8, 8.20
Rotor Seal 6, 3.3, 7.5, 7.6
Running a Chromatogram 3.14
Safety Features 2.13

Sample Chromatogram 6.2, 6.2-4, 6.2-6, 6.3, 6.3-5, 6.4, 6.4-5
Sample Preparation 6.2, 6.2-2, 6.3, 6.3-2, 6.3-3, 6.4, 6.4-1, 7.5, 8.22
Seal Replacement 2.9, 5.12, 5.14
Secondary Amines 6.2-1, 6.2-2, 6.2-19
Sequence 1, 3.14, 3.15, 4.1, 4.3, 4.4, 4.5, 4.7, 4.9, 4.10, 4.11, 4.13, 4.14, 4.15, 4.16, 4.17, 4.18,
5.4, 5.5, 5.12, 7.8, 7.9, 8.4, 8.5
SERAPREP 6.2-3, 8.22
Shelf Life 6.2-19, 8.21
Shipping 4.5, 5.6
Shutdown 2.3, 2.5, 3.15, 4.11, 4.17, 4.18, 5.2, 5.3, 5.4, 5.5, 5.8, 5.12, 5.16, 6.2-18, 6.3-12, 6.4-6,
7.4, 7.6
Silica 5.3, 7.4, 7.11, 7.12
Site Requirements 1, 4, 2.12, 3.2, 8.2
Sodium Hypochlorite 6, 7, 6.2-5, 6.4-2
Sodium ion-exchange 6.2, 8.21
Software 4, 5, 2.3, 3.4, 3.6, 3.13, 3.14, 3.15, 4.2, 4.4, 4.5, 4.6, 4.7, 4.9, 4.10, 4.11, 4.12, 4.13,
4.14, 4.15, 4.16, 4.17, 5.4, 5.5, 5.6, 5.9, 5.12, 7.2, 7.8, 7.9, 7.10, 8.3
Spare Parts 8.21
Standard Configurations 2.14
Standards 6, 3.1, 6.2, 6.3-3, 7.5, 8.22
Stop/Reset 4.5, 4.17
Storage 3.10, 3.15, 3.16, 4.5, 4.9, 4.18, 5.1, 5.4, 5.5, 5.6, 6.3-3, 7.5
Symbols and Warnings 2
Index
T
Technical Support 7.1, 7.6

Tefzel 6, 3.3, 6.2, 8.19
Thiofluor 6, 5.3, 6.2-2, 6.2-4, 6.2-5, 6.2-19, 6.3-2, 6.3-4, 6.3-5, 6.3-12, 6.4-1, 6.4-3, 6.4-4, 6.4-6,
8.9, 8.10, 8.21, 8.23
Trione 7, 3.2, 3.3, 3.14, 6.2, 6.2-3, 6.2-4, 6.2-19, 7.4, 7.5, 7.7, 8.11, 8.13, 8.21
Troubleshooting 1, 6.3-2, 7.2, 7.3, 7.7, 7.8, 7.9, 7.10, 8.25
Tutorials 4.11
URIPREP 6.2-3, 8.22

USB 3, 5, 2.11, 3.4, 3.6, 4.1, 4.6, 7.9, 8.20
Valve Maintenance 5.9, 5.10, 8.19

Valve Parts 8.19
Vegetable Samples 6.4, 6.4-1
Warranty 5.1, 5.2, 5.3, 6.2-19, 6.3-4, 6.3-12, 6.4-3, 6.4-6, 8.24
Water Samples 3.3, 3.4, 6.3-2, 6.4-1, 6.4-2, 7.4

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Operator Manual

1280 Space Park Way

Cat. No. 0101-0008

Version 10, April 2016

What is Post-column Derivatization? 1.1

Pinnacle Operators Manual

Site Requirements 3.2

Pump and Autosampler Connections 3.8

Pinnacle PCX Operation

Pinnacle Operators Manual

Maintenance Suggestions 5.1

Amino Acids 6.2

Pinnacle Operators Manual

Contact Pickering Laboratories For Support

Pinnacle Operators Manual

Pinnacle Operators Manual

Pinnacle Operators Manual

How to Use this Manual

How to Use this Manual

Pinnacle Operators Manual

Read this First!

Symbols and Warnings

Pinnacle Operators Manual

Pinnacle Operators Manual

Radio Frequency Pinnacle PCX complies with IEC 61000-4-3

Minimum 3 inches clearance at back of instrument for venting.

Pinnacle Operators Manual

Pinnacle Operators Manual

Pinnacle Operators Manual

For Amino Acid Analysis

Pinnacle Operators Manual

Pinnacle Operators Manual

What is Post-column Derivatization?

Requirements for a Successful Post-column Method

Pinnacle Operators Manual

Design of a HPLC system

Pinnacle Operators Manual

Pinnacle Operators Manual

Designing a Post-column System

Pinnacle Operators Manual

Pinnacle Operators Manual

Design of the Pinnacle PCX

Pinnacle Operators Manual

Pinnacle Operators Manual

In addition to accomplishing the above three tasks, the

This chapter is designed to familiarize you with the

Flow Path Dual Pump

Flow Path Single Pump

Pinnacle Operators Manual

The flow path of the Pinnacle PCX is extremely inert,

The column check valve is located just outside of the

Column outlet tubing

Column Connections and Column Oven

The heater block is designed to receive the analytical

Pinnacle Operators Manual

Pinnacle Operators Manual

The valves are connected to a Flush Bottle which

Reservoir Tray / Bottles

There are 4 bottles located here:

Pinnacle Operators Manual

Reagent tubing is pre-connected to the pump at the factory. All

Pinnacle Operators Manual

Pinnacle Operators Manual

Quick Change Reactor